(L.) Spreng. Fruits: Antimicrobial Efficiency and their Phytochemical Constituents

Antimicrobial analysis of hexane, chloroform, methanol and aqueous extracts of method and minimum inhibitory concentration was determined by serial two species including Gram positive and Gram negative bacteria and three fungal species were used the Gram positive bacteria and fungi were more sensitive than the Gram negative bacteria, to both solvent and aqueous extracts. Moreover, resistant to the tested extracts. Phytochemical analysis of fruits revealed the presence of secondary metabolites like alkalo terpenoids, which have been implicated in Bridelia retusa

Spreng. (family Euphorbiaceae to moderate sized deciduous tree, found in India, Bangladesh, Nepal, Sri Lanka, Southern China, Indochina and Sumatra. Traditionally, it is valuable as astringent, used in rheumatism problems, urinary infections; the plant promote antifertiliy and wound healing. Stem bark is used to treat dysentery, diarrhea and diabetes. Leaves and fruits are used as antifungal and for stomach ache (Mishra and Sahu, 1984;Nadkarni and Nadkarni, 2000;Jayasinghe et al., 2003). These different operties are due to the presence of different chemical constituents as isoflavone (Adhavet, 1998), decanoic acie, stigmasterol, dehydrostigmasterol, β tannins and triterpenes. Fruit pulp contains gallic acid, ellagic otra and Moorthy, 1973). It is well known also for the presence of tannins. It is reported to be used traditionally in snake bites, wounds and tonics for et al., 1980). Phenolics, including tannins, are the natural products prese amount and possess various biological properties related to anti inflammatory effects (Mehare and Hatapkki, 2003), wound healing activity (Bagad, 2007), antioxidant (Tatiya ., 2011), antimicrobial activity of stem bark (Tatiya ., 2012) or leaves (Khan and Khan, 2013). Various parts of the plants were reported for antimicrobial activity, but there are no reports on antimicrobial activity of fruits, hence the present investigation was carried out. Thus the antimicrobial activities solvent and aqueous extracts of B. retusa fruits were analyzed. Antimicrobial analysis of hexane, chloroform, methanol and aqueous extracts of method and minimum inhibitory concentration was determined by serial two species including Gram positive and Gram negative bacteria and three fungal species were used the Gram positive bacteria and fungi were more sensitive than the Gram negative bacteria, to both solvent and aqueous was found as the most sensitiv resistant to the tested extracts. Phytochemical analysis of fruits revealed the presence of secondary metabolites like alkalo antimicrobial activities. Hence, it would be recommended to explore the maximum potential of in the medicinal and pharmaceutical field and investigation are endorsed for further application useful in phytomedicine.

Bridelia retusa, fruit extracts, phytochemical
Available online: www.notulaebiologicae.ro Print ISSN 2067Not Sci Biol, 2016 Spreng. (family Euphorbiaceae) is a small to moderate sized deciduous tree, found in India, Bangladesh, Nepal, Sri Lanka, Southern China, Indochina and Sumatra. Traditionally, it is valuable as astringent, used in rheumatism problems, urinary infections; the plant promote antifertiliy and wound healing. Stem bark is used to treat dysentery, diarrhea and diabetes. Leaves and fruits are used as antifungal and for stomach ache (Mishra and Sahu, 1984;Nadkarni and ., 2003). These different operties are due to the presence of different chemical constituents as isoflavone (Adhavet, 1998), decanoic acie, stigmasterol, dehydrostigmasterol, β-sitosterol, tannins and triterpenes. Fruit pulp contains gallic acid, ellagic otra and Moorthy, 1973). It is well known also for the presence of tannins. It is reported to be used traditionally in snake bites, wounds and tonics for ., 1980). Phenolics, including tannins, are the natural products present in abundant amount and possess various biological properties related to anti inflammatory effects (Mehare and Hatapkki, 2003), wound healing activity (Bagad, 2007), antioxidant (Tatiya ., 2011), antimicrobial activity of stem bark (Tatiya ., 2012) or leaves (Khan and Khan, 2013). Various parts of the plants were reported for antimicrobial activity, but there are no reports on antimicrobial activity of fruits, hence the present investigation was carried out. Thus the antimicrobial activities of both fruits were analyzed. Antimicrobial analysis of hexane, chloroform, methanol and aqueous extracts of method and minimum inhibitory concentration was determined by serial two species including Gram positive and Gram negative bacteria and three fungal species were used the Gram positive bacteria and fungi were more sensitive than the Gram negative bacteria, to both solvent and aqueous was found as the most sensitive bacteria, whereas resistant to the tested extracts. Phytochemical analysis of fruits revealed the presence of secondary metabolites like alkalo antimicrobial activities. Hence, it would be recommended to explore the maximum potential of in the medicinal and pharmaceutical field and investigation are endorsed for further application useful in phytomedicine.
, fruit extracts, phytochemical www.notulaebiologicae.ro Print ISSN 2067-3205;Electronic 2067Not Sci Biol, 2016 ) is a small to moderate sized deciduous tree, found in India, Bangladesh, Nepal, Sri Lanka, Southern China, Indochina and Sumatra. Traditionally, it is valuable as astringent, used in rheumatism problems, urinary infections; the plant promote antifertiliy and wound healing. Stem bark is used to treat dysentery, diarrhea and diabetes. Leaves and fruits are used as antifungal and for stomach ache (Mishra and Sahu, 1984;Nadkarni and ., 2003). These different operties are due to the presence of different chemical constituents as isoflavone (Adhavet, 1998), sitosterol, tannins and triterpenes. Fruit pulp contains gallic acid, ellagic otra and Moorthy, 1973). It is well known also for the presence of tannins. It is reported to be used traditionally in snake bites, wounds and tonics for ., 1980). Phenolics, including nt in abundant amount and possess various biological properties related to anti inflammatory effects (Mehare and Hatapkki, 2003), wound healing activity (Bagad, 2007), antioxidant (Tatiya et ., 2011), antimicrobial activity of stem bark (Tatiya et al., ., 2012) or leaves (Khan and Khan, 2013). Various parts of the plants were reported for antimicrobial activity, but there are no reports on antimicrobial activity of fruits, hence the present investigation of both fruits were analyzed.

Collection, identification and extraction Bridelia retusa
Kambalakonda forest area, Visakhapatnam, Andhra Pradesh, India. Department of Botany, Andhra University, Visakhapatnam, India. The collected fruits were dried in the shadow until completely dried. Then the dried fruits were powdered in the mixture grinder and p extraction was done using hexane, chloroform, followed by methanol. The filtrates were concentrated by removing the solvents under reduced pressure, at 40 °C, using a rotary evaporator. The concentrated crude extracts stored at 4 °C.
Simultaneously, the aqueous extract of the fruits was prepared by adding boiled water to the powdered fruits in a beaker on water bath, with occasional stirring for 4 hrs. The aqueous extract was then filtered and reduced

Bacterial and fungal strains used
The strains used within the experiment were procured from the Microbial Type Culture and Collection ( Antimicrobial analysis of hexane, chloroform, methanol and aqueous extracts of Bridelia retusa method and minimum inhibitory concentration was determined by serial two-fold dilution method. Seven human pathogenic bacteria species including Gram positive and Gram negative bacteria and three fungal species were used the Gram positive bacteria and fungi were more sensitive than the Gram negative bacteria, to both solvent and aqueous e bacteria, whereas Proteus vulgaris resistant to the tested extracts. Phytochemical analysis of fruits revealed the presence of secondary metabolites like alkalo antimicrobial activities. Hence, it would be recommended to explore the maximum potential of in the medicinal and pharmaceutical field and investigation are endorsed for further application useful in phytomedicine.

Collection, identification and extraction Bridelia retusa
Kambalakonda forest area, Visakhapatnam, Andhra Pradesh, India. The collected fruits were identified by Prof. M. Venkaiah, Department of Botany, Andhra University, Visakhapatnam, India. The collected fruits were dried in the shadow until completely dried. Then the dried fruits were powdered in the mixture grinder and p extraction was done using hexane, chloroform, followed by methanol. The filtrates were concentrated by removing the solvents under reduced pressure, at 40 °C, using a rotary evaporator. The concentrated crude extracts stored at 4 °C.
Simultaneously, the aqueous extract of the fruits was prepared by adding boiled water to the powdered fruits in a beaker on water bath, with occasional stirring for 4 hrs. The aqueous extract was then filtered and reduced Bridelia retusa fruits was performed by agar well fold dilution method. Seven human pathogenic bacteria species including Gram positive and Gram negative bacteria and three fungal species were used in the study and the results indicated that the Gram positive bacteria and fungi were more sensitive than the Gram negative bacteria, to both solvent and aqueous Proteus vulgaris and resistant to the tested extracts. Phytochemical analysis of fruits revealed the presence of secondary metabolites like alkalo antimicrobial activities. Hence, it would be recommended to explore the maximum potential of in the medicinal and pharmaceutical field and investigation are endorsed for further application useful in phytomedicine.

Material and Methods
Collection, identification and extraction Bridelia retusa (L.) Spreng. fruits were collected from Kambalakonda forest area, Visakhapatnam, Andhra Pradesh, The collected fruits were identified by Prof. M. Venkaiah, Department of Botany, Andhra University, Visakhapatnam, India. The collected fruits were dried in the shadow until completely dried. Then the dried fruits were powdered in the mixture grinder and packed in Soxhlet apparatus. Sequential extraction was done using hexane, chloroform, followed by methanol. The filtrates were concentrated by removing the solvents under reduced pressure, at 40 °C, using a rotary evaporator. The concentrated crude extracts Simultaneously, the aqueous extract of the fruits was prepared by adding boiled water to the powdered fruits in a beaker on water bath, with occasional stirring for 4 hrs. The aqueous extract was then filtered and reduced fruits was performed by agar well fold dilution method. Seven human pathogenic bacteria in the study and the results indicated that the Gram positive bacteria and fungi were more sensitive than the Gram negative bacteria, to both solvent and aqueous Bridelia retusa and Aspergillu niger resistant to the tested extracts. Phytochemical analysis of fruits revealed the presence of secondary metabolites like alkaloid, saponins and antimicrobial activities. Hence, it would be recommended to explore the maximum potential of in the medicinal and pharmaceutical field and investigation are endorsed for further application useful in phytomedicine.

Collection, identification and extraction
(L.) Spreng. fruits were collected from Kambalakonda forest area, Visakhapatnam, Andhra Pradesh, The collected fruits were identified by Prof. M. Venkaiah, Department of Botany, Andhra University, Visakhapatnam, India. The collected fruits were dried in the shadow until completely dried. Then the dried fruits were powdered in the acked in Soxhlet apparatus. Sequential extraction was done using hexane, chloroform, followed by methanol. The filtrates were concentrated by removing the solvents under reduced pressure, at 40 °C, using a rotary evaporator. The concentrated crude extracts were labeled and Simultaneously, the aqueous extract of the fruits was prepared by adding boiled water to the powdered fruits in a beaker on water bath, with occasional stirring for 4 hrs. The aqueous extract was then filtered and reduced under pressure. in the study and the results indicated that Bridelia retusa fruit Aspergillu niger were the most id, saponins and antimicrobial activities. Hence, it would be recommended to explore the maximum potential of in the medicinal and pharmaceutical field and investigation are endorsed for further application useful in phytomedicine.

Bacterial and fungal strains used
(L.) Spreng. fruits were collected from Kambalakonda forest area, Visakhapatnam, Andhra Pradesh, The collected fruits were identified by Prof. M. Venkaiah, Department of Botany, Andhra University, Visakhapatnam, India. The collected fruits were dried in the shadow until completely dried. Then the dried fruits were powdered in the acked in Soxhlet apparatus. Sequential extraction was done using hexane, chloroform, followed by methanol. The filtrates were concentrated by removing the solvents under reduced pressure, at 40 °C, using a rotary were labeled and Simultaneously, the aqueous extract of the fruits was prepared by adding boiled water to the powdered fruits in a beaker on water bath, with occasional stirring for 4 hrs. The under pressure. fruits was performed by agar well fold dilution method. Seven human pathogenic bacteria in the study and the results indicated that fruit were the most id, saponins and antimicrobial activities. Hence, it would be recommended to explore the maximum potential of (L.) Spreng. fruits were collected from Kambalakonda forest area, Visakhapatnam, Andhra Pradesh, The collected fruits were identified by Prof. M. Venkaiah, Department of Botany, Andhra University, Visakhapatnam, India. The collected fruits were dried in the shadow until completely dried. Then the dried fruits were powdered in the acked in Soxhlet apparatus. Sequential extraction was done using hexane, chloroform, followed by methanol. The filtrates were concentrated by removing the solvents under reduced pressure, at 40 °C, using a rotary were labeled and Simultaneously, the aqueous extract of the fruits was prepared by adding boiled water to the powdered fruits in a beaker on water bath, with occasional stirring for 4 hrs. The The strains used within the experiment were procured from the Microbial Type Culture and Collection (

Antimicrobial efficiency
The lyophilized culture was sub-cultured and concentration of working stock culture was assessed as 10 -6 CFU/ml. Specified quantity of nutrient agar was prepared and plated in aseptic conditions. The agar well diffusion technique was performed for antimicrobial susceptibility test for crude extracts and dimethyl sulfoxide (DMSO), whereas agar disc diffusion method was followed for antimicrobial susceptibility test for standard antibiotic discs. The extracts were dissolved in DMSO to get the known concentrations of 25 mg/ml, 50 mg/ml and 100 mg/ml respectively. After 24 h of incubation at 37 °C, the zone of inhibition was measured using an antibiotic zone reader scale (HiAntibiotic ZoneScale-c) and tabulated. For the antifungal activity, the same method as for bacteria, of nutrient agar respectively, was adopted, whereas Saboraud dextrose agar was used. The inoculated medium was incubated at 25 °C for two days for the C. albicans, and S. cerevisiae and three days for A. niger (Aniel Kumar et al., 2014). About 500 µg of nystatin was dissolved in 1 ml of sterile de-ionized water. About 10 µl of 0.5 mg/ml nystatin (equivalent to 5 µg dose) and 10 µl of DMSO was pipetted into wells. For bacteria, multidrug antibiotic disc was used (Axiom Laboratories Ltd., India). The experiments were conducted in triplicates each and diameter of the inhibition zone surrounding each well was recorded and tabulated. 34 The extracts that exhibited inhibition zones were subjected to minimum inhibition concentration (MIC) assay by using serial two-fold dilution method (Aniel Kumar et al., 2010). A quantity of 0.6 g of each extract was dissolved in 300 ml sterile nutrient broth which yielded initial concentration of 2,000 µg/ml. Subsequently, serial dilution was made from the stock to obtain 1,000; 500; 250; 125; 62.5; 31.2 µg/ml concentrations. One ml of standardized inoculums of each test organism was introduced into each extract, nutrient broth mixture and then incubated at 37 °C. The lowest concentration inhibiting growth of microorganism was regarded as the MIC of the extracts. For the fungi, the inoculated medium was incubated at 25 °C for two (C. albicans, S. cerevisiae) to three (A. niger) days.

Statistical analysis
Each experimental data from triplicates was subjected to one way ANOVA using Minitab version 15. A significant level of p < 0.01 was used for all statistical analyses.

Results and Discussion
The antimicrobial activity of fruit extracts of B. retusa was assayed by agar well diffusion method against seven bacterial strains including Gram positive B. subtilis, E. faecalis, M. luteus, S. aureus, S. pneumoniae, Gram negative E. coli, K. pneumoniae, P. aeruginosa, P. vulgaris, fungi A. niger, C. albicans and S. cerevisiae. Table 1 shows the microbial growth inhibition zones   albicans. E. faecalis exhibited inhibition zones similar or larger than standard antibiotic tetracycline, while B. subtilis, S. pneumoniae, C. albicans gave better results for chloroform, methanol and aqueous extracts; S. aureus had a larger inhibition zone in the case of methanol extract. Methanol and aqueous extracts also showed high inhibition zones against fungal strains C. albicans and S. cerevisiae. Although the Gram negative bacteria were sensitive for all extracts, did not show a broad spectrum of antimicrobial activity. The negative control of DMSO had no effect on the microbial growth of all tested bacteria and fungi. Hence, the absence of inhibition zones confirmed that DMSO could not act as antimicrobial agent.
The effect of different solvents such as water, ethanol (50%), methanol (50%) and acetone (70%) of B. retusa stem bark exhibited antimicrobial activity against Gram positive bacteria B. subtilis, S. aureus and Gram negative bacteria E. coli and fungi C. albicans (Tatiya et al., 2011), whereas in the present study B. retusa fruits extracts had also shown high antimicrobial activity against Gram positive bacteria B. subtilis, E. faecalis, S. pneumoniae and fungi C. albicans.  (Tatiya et al., 2011).
Phytochemical analysis (Table 3) revealed that B. retusa fruits posses alkaloids, aminoacids, anthraquinone, carbohydrates, cardiac glycosides, flavonoids, glycosides, phenols, saponins, steroids, tannins, terpenoids and volatile compounds. Methanol extract exhibited positive results for all tested phytochemicals, whereas the observed antimicrobial activity may be due to the presence of some metabolites like alkaloid, saponins and terpenoids, which are implicated in various biological activities (Thomas et al., 2013). The presence of these metabolites suggests great potential for the plant as a source of useful phytomedicines (Kunle et al., 2003).

Conclusions
It may be concluded that the results of the present study support the folkloric usage of the B. retusa as a medicine. The results indicated that the Gram positive bacteria and fungi were more sensitive than the Gram negative bacteria, to both solvent and aqueous Bridelia retusa fruit extracts. Phytochemical analysis revealed that B. retusa fruits posses alkaloids, aminoacids, anthraquinone, carbohydrates, cardiac glycosides, flavonoids, glycosides, phenols, saponins, steroids, tannins, terpenoids and volatile compounds; antimicrobial activity may be due to the presence of some metabolites like alkaloid, saponins and terpenoids, which are implicated in various biological activities. Hence, it is necessary to explore the maximum potential of the plant in medicinal field and pharmaceutical sciences for further application. Further studies are required about the appropriate characterization of the compounds present in the B. retusa fruits.