Phytochemical and Antimicrobial Analysis of Callus Extracts of Biophytum sensitivum (Linn) DC

Aims: In vitro studies are highly instrumental in selecting a drug for a particular disease and also in getting the preliminary evidence to proceed for further In vivo pharmacological research. Hence, the study is designed to screen and identify the therapeutic suitability of this plant extract for the treatment of a particular disease. And to find out the presence of phytochemicals and antimicrobial activity of leaf callus cultures of Biophytum sensitivum Linn. Place and Duration of Study: Department of Botany and Microbiology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur 522510, India during June 2010 to Dec 2010. Methodology: Here we induced the callus from the leaf explants of this species on Murashige and Skoog basal medium supplemented with various concentrations of BA and NAA. BA 1.0 mg/l with NAA 1.0mg/l is the best concentration for optimal results. The callus was extracted sequentially with hexane, chloroform, ethyl acetate and methanol for Original Research Article British Microbiology Research Journal, 4(8): 869-884, 2014 870 24h by using Soxhlet apparatus. These extracts were used to investigate the presence of phytochemicals which was performed according to the Aiyelaagbe and Osamudiamen [29] and Egwaikhide et al. [30] methods. The mean values were statistically analyzed with the MINITAB 14 by the general one way (un stacked) analysis of variance (ANOVA) to find out the most effective extracts Results: The qualitative phytochemical analysis of various solvent extracts showed the presence of phytochemicals viz., Terpenoids, phenols, flavonoids, saponins, quinones and phenols. All the extracts except hexane showed highest zone of inhibition against gram positive and gram negative bacteria (4.46-22.9mm) as well as fungi (7.64-144.4mm) by agar well diffusion method at 100ppm concentrations. The results of present study indicate that the callus of this plant is a potential source of antimicrobial agents and drugs and need to be investigated further. Conclusion: From the present study, it is evident that, the antibacterial active constituent of Biophytum sensitivum is having a constant expression pattern over different pathogens. This plant leaf callus can be further subjected to enhancement and isolation of the therapeutic antimicrobials and carry out further pharmacological evaluation.


INTRODUCTION
Since their discovery during 20th century, antimicrobial agents have substantially reduced the threat posed by infectious diseases. The use of these "wonder drugs" has led to a dramatic drop in deaths from diseases that were previously wide spread, untreatable and frequently fatal. These drugs have also contributed to the major gains in life expectancy. The bacterial infections which contribute mostly to human diseases such as acquired infections are also developing resistance against drugs used to treat them [1]. The treatment of infectious diseases still remains as an unsolved global problem because of newly emerging diseases and increasing number of multi-drug resistant Gram-positive and Gram-negative bacteria. The usage of antimicrobials by human has been increased from 100 to 1000 times.
The most important bioactive constituents of plants are terpenoids, alkaloids, tannins and flavonoid compounds [2]. The presence of these secondary metabolites could be responsible for some of the observed antimicrobial activity. Plant based antimicrobial compounds serve without side effects than synthetic compounds and has enormous therapeutic potential. Although very few plant cell processes are operating commercially, the most successful commercial pharmaceuticals produced from undifferentiated cell cultures are antibiotic compounds [3].
There has been increasing interest in the production of secondary metabolites using cell and callus cultures for commercial use by many medicinal plants [4,5,6,7,8,9]. Cell and callus cultures represent the best source of cell mass for production of secondary metabolites due to unique environmental stress [2,3]. In vitro studies are highly instrumental in selecting a drug for a particular disease and also in getting the preliminary evidence to proceed for further In vivo pharmacological research. Hence, the study is designed to screen and identify the therapeutic suitability of this plant extract for the treatment of a particular disease.
Biophytum sensitivum Linn. DC. (Syn. Oxalis sensitiva Linn.) belongs to the family Oxalidaceae is a small, sensitive annual herb found throughout the tropical regions of South Asia, Africa and Madagaskar. It is well known in Andhra Pradesh, India with its vernacular name as Jalapushpa, Attapatti and used as a folk medicine against diabetes. It grows in shady places in dry parts of India during the rainy season. It is extensively used in traditional oriental herbal medicines [10].
B. sensitivum is widely used in Ayurveda and Siddha systems of Indian traditional medicine to treat various ailments. The pharmacological studies of this plant have reported antioxidant, antibacterial, antitumor, antimetastatic, antiangiogenesis, antidiabetic, anticancer, antiinflammatory, immunomodulatory, chemoprotective, cardioprotective, radioprotective and wound healing properties. Phytochemical analysis of this species has reported important medicinal compounds such as amentoflavone, cupressuflavone and isoorientin. Recently this species has gained enormous pharmacological importance.
Flowering occurs during August to January and due to seed dormancy, the cultivation of this plant has become very difficult. It thrives only on slightly acidic and damp soil. Shivanna et al. [10] in 2009 established a protocol for In vitro regeneration of B. sensitivum. Owing to the great medicinal importance of this species and due to difficulty in propagation, in our current study we induced the calli of B. sensitivum and carried out solvent extraction of In vitro callus with increasing polarity viz., hexane, chloroform, ethyl acetate and methanol. These extracts were collected and used for phytochemical and antimicrobial study.

Plant Material
The fresh matured 100 plants of B. sensitivum were collected from Acharya Nagarjuna University Campus, Guntur District, Andhra Pradesh, India during June 2010 to Dec 2010 and used as a source of explants. The leaf explants were excised into 1 cm long segments and were washed with liquid detergent (Teepol, Qualigens, India), Bavistin (1% w/v) for 3 min and Mercuric Chloride (0.1% w/v) for 1min, followed by 70% ethanol. These are inoculated on Murashige and Skoogs medium [28] supplemented with various concentrations and combinations of phyto hormones for induction of callus. The combination of NAA+BA (BA 0.5-5.0mg/l, NAA 0.5-5.0mg/l) produced callus.

Callus Culture
The leaf explants were cultured on M.S. basal medium supplemented with various concentrations of BA+NAA for callus induction. BA 1.0mg/l + NAA 1.0mg/l is the best concentration for callus induction. After 30-60 days, old callus was collected and sub cultured on fresh medium with same growth regulator combinations twice in four week time interval. All the cultures were incubated at 24±2ºC under 16h photoperiod provided by cool white florescent lights.

Extraction from Callus Cultures
About 6-8 week-old calli ( Fig. 1) derived from the leaf cuttings were collected and dried in an oven at 40±1ºC for 5h. Dried calli was homogenized to a fine powder and stored in airtight bottles. 25g of shoot calli powder was extracted with 150ml solvent of each of hexane, chloroform, ethyl acetate and methanol for 24h by using Soxhlet apparatus. All the extracts were concentrated separately on rotary evaporator at 40ºC. 100mg/ml were prepared by redissolving the extracted powder in the same solvent which was used for the extraction. This crude callus extract is used for qualitative phytochemical analysis and anti-microbial activity.

Phytochemical Screening
The preliminary phytochemical analysis was performed according to the Aiyelaagbe et al. [29] and Egwaikhide et al. [30] methods.

Antimicrobial Assay
The antimicrobial activity of the crude extracts of B. sensitivum was determined by agar well diffusion method of Cappuccino and Sherman [31] and Volk et al. [32]. Nutrient agar (NA) and Czapek-Dox (CD) agar media were used for culturing the test bacteria and fungi respectively. NA medium (100ml) was sterilized at 15 lbs pressure (12ºC) for 15min, cooled and inoculated with 0.2ml of test bacterial suspension. After thorough mixing, the seed medium was poured into Petri plates under aseptic conditions. After solidification of agar medium, wells of about 4 mm diameter were punched into it with sterilized cork borer. In case of antifungal assay, spore suspension of test fungus (10 5 Spores/ml) was mixed with the cooled, molten CD agar medium and poured into Petri dishes. Wells were made in the medium after solidification. The crude extract dissolved in hexane, chloroform, ethyl acetate and methanol at a concentration of 25, 50, 75 and 100ppm was added to each well. Streptomycin served as positive control while hexane, chloroform, ethyl acetate and methanol served as negative control for both bacterial and fungal strains. Fluconazole (0.1ppm/well) served as the positive experimental control for all fungi strains assayed. The plates were incubated at 37ºC for 24h for bacteria, 24-72h for yeast and filamentous fungi and the diameter of the inhibition zones was measured.

Statistical Analysis
The mean values were statistically analyzed with the MINITAB 14 by the general one way (un stacked) analysis of variance (ANOVA) to find out the most effective extracts and the most sensitive test organisms. Differences between antimicrobial assays by ANOVA followed by LSD (P<0.05).

Antimicrobial Activity
From     ANOVA data on the antimicrobial activity of chloroform, ethyl acetate and methanol extracts had significant effect (P<0.05) on the levels of concentrations of extracts. The results showed that the studied plant callus extracts were potentially a rich source of antimicrobial agents. However the plants differ significantly in their activity against the test microorganisms. According to one way ANOVA results, antimicrobial activity has also shown differences among the taxa (P=0.0042, F=4.62, R=0.1). Hexane, chloroform, ethyl acetate and methanol were used as negative control for which a number of few inhibitory effects could be observed.
In all these cases, the increased zone of inhibition is observed in the methanolic extracts. But ethyl acetate extract also showed high zone of inhibition in B. subtilis (19.12mm) (Plate 1B), indicating that ethyl acetate extracts contains specific phytochemicals needed for inhibition of growth of these bacteria. When the zone of inhibition of gram positive bacteria are compared with gram negative bacteria, increased zone of inhibition was observed with later indicating that the callus extracts of B. sensitivum contain highly important antibiotics. The qualitative phytochemicals analysis confirms the above observation (Table 1). ANOVA data on the antimicrobial activity of chloroform, ethyl acetate and methanol extracts had significant effect (P<0.05) on the levels of concentrations of extracts. The results showed that the studied plant callus extracts were potentially a rich source of antimicrobial agents. However the plants differ significantly in their activity against the test microorganisms. According to one way ANOVA results, antimicrobial activity has also shown differences among the taxa (P=0.0042, F=4.62, R=0.1). Hexane, chloroform, ethyl acetate and methanol were used as negative control for which a number of few inhibitory effects could be observed.

I) Salmonella typhi
In all these cases, the increased zone of inhibition is observed in the methanolic extracts. But ethyl acetate extract also showed high zone of inhibition in B. subtilis (19.12mm) (Plate 1B), indicating that ethyl acetate extracts contains specific phytochemicals needed for inhibition of growth of these bacteria. When the zone of inhibition of gram positive bacteria are compared with gram negative bacteria, increased zone of inhibition was observed with later indicating that the callus extracts of B. sensitivum contain highly important antibiotics. The qualitative phytochemicals analysis confirms the above observation (Table 1). ANOVA data on the antimicrobial activity of chloroform, ethyl acetate and methanol extracts had significant effect (P<0.05) on the levels of concentrations of extracts. The results showed that the studied plant callus extracts were potentially a rich source of antimicrobial agents. However the plants differ significantly in their activity against the test microorganisms. According to one way ANOVA results, antimicrobial activity has also shown differences among the taxa (P=0.0042, F=4.62, R=0.1). Hexane, chloroform, ethyl acetate and methanol were used as negative control for which a number of few inhibitory effects could be observed.
In all these cases, the increased zone of inhibition is observed in the methanolic extracts. But ethyl acetate extract also showed high zone of inhibition in B. subtilis (19.12mm) (Plate 1B), indicating that ethyl acetate extracts contains specific phytochemicals needed for inhibition of growth of these bacteria. When the zone of inhibition of gram positive bacteria are compared with gram negative bacteria, increased zone of inhibition was observed with later indicating that the callus extracts of B. sensitivum contain highly important antibiotics. The qualitative phytochemicals analysis confirms the above observation (Table 1).
Methanolic extracts showed potent activity against all tested bacteria which could be due to various secondary metabolites like flavonoids, saponins, terpenoids and phenols as noticed in qualitative analysis of extracts (Table 1). Hence the methanol was noticed to be the best solvent among the three.

Antifungal Activity
Like in the case of antibacterial activity, anti fungal activity is also exhibited by various extracts of B.sensitivum (Table 3). Extracts inhibited the growth of six fungal species and is ineffective against two species namely, Fusarium solani and Fusarium oxysporum ( In 2010, Natarajan et al. [26] reported the antibacterial activity of wild plant leaf extracts of B.sensitivum with petroleum ether, chloroform, acetone and methanol. It showed potent antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, S. pneumoniae, Klebsiella pneumoniae, Salmonella typhi, Proteus vulgaris and Escherichia coli by agar well diffusion method. All the extracts inhibited the growth of almost all the selected bacteria in the range of 7-25 mm. Among these, acetone extract showed great antibacterial activity, chloroform and methanol extracts showed better activity than petroleum ether extract.
Ribona et al. [33] in 2011 reported the antimicrobial activity of aqueous, ethanolic and acetone extracts of B.sensitivum on pathogens like E. coli, Klebsiella and Proteus by agar well diffusion method. In case of Klebsiella, Proteus and E. coli the inhibition zone was 9 mm for aqueous extracts in maximum concentrations. However in case of Klebsiella and Proteus inhibition zone of 11mm for ethanolic extracts was observed. Whereas E. coli and Proteus showed 9 mm zone of inhibition which was more effective when compared to acetone and aqueous extracts. Inhibition of growth of both gram positive and gram negative bacteria by these extracts indicates the potentiality for broad spectrum activity. The antibacterial potential activity of methanolic and chloroform extracts in other plant systems has been reported [34]. Other species from Oxalidaceae family were also showed good antimicrobial activities. Oxalis corniculata leaf extract showed antibacterial activity against several human pathogenic bacteria [7,35]. It was reported that stem bark extracts of Averrhoa carambola showed potent antibacterial and antifungal activities [36], antibacterial and cytotoxic activities using this plant fruit extract [37] and antioxidant activities on fruit at various stages of ripening [38].
The data pertaining to the antibacterial and antifungal potential of the callus culture explants of B. sensitivum were presented in Tables 2 and 3. The extracts from callus culture were obtained from two different hormonal combinations. Previously reported that BA 1mg/l and NAA 1mg/l is best concentration for leaf callus induction of B. sensitivum [39,40]. The results of the present study revealed the positive relation between production of active metabolites in the callus and growth regulator (NAA) in the media. The activity can be positively correlated to the dose, as there is an increase in the zone of inhibition with increased dose. All the extracts except hexane had good and similar activities against bacteria and ethyl acetate and methanol extracts showed good activity for fungal human pathogens. As chloroform, ethyl acetate and methanol are polar solvents than non polar hexane, extracts prepared by methanol provided more consistent antimicrobial activity. This might have resulted from the best of solubility of the active constituents in methanol extract.
Such screening of various natural organic compounds and identifying active agents is the need of the hour, because successful prediction of a lead molecule and drug like properties at the onset of drug discovery will pay off later in drug development. From the present study it is evident that, the antibacterial active constituent of B. sensitivum is having a constant expression pattern over different pathogens. This plant leaf callus can be further subjected to enhancement and isolation of the therapeutic antimicrobial and carry out further pharmacological evaluation. It is highly valuable species, with impressive range of medicinal uses. Further work is mandatory to isolate the active principle from the callus extracts and to carry out pharmacological studies.

CONCLUSION
It is a highly valuable species with wide range of antimicrobial activities. Further work is mandatory for isolation and identification of pure active antibacterial and antifungal constituents from the callus extracts and to carry out pharmacological studies.