PHYTOCHEMICAL ANALYSIS AND EVALUATION OF ANTIMICROBIAL ACTIVITY IN THE WHOLE PLANT EXTRACTS OF GLORIOSA SUPERBA

Objectives: The aim of this research work was to carry out the phytochemical screening and to evaluate the antibacterial and antifungal activity of the whole plant extract (shoot, flower, and tuber) of the flame lily (Gloriosa superba). Materials and Methods: In the current work, phytochemicals were extracted from different parts of the plant using different solvents dimethyl sulfoxide, ethyl acetate, and ethanol (ETOH). These phytochemicals contained alkaloids, flavonoids, terpenoids, tannins, saponins, aromatic acids, phenolic compounds, xanthoproteins, triterpenoids, amino acids, philobatinins, carbohydrate, reducing sugar, and proteins and they were separated using standard methods. Furthermore, antimicrobial activities of methanolic separation were determined using various species of bacteria and fungi. Agar well diffusion method was used for the antimicrobial activity and the zone of the inhibition was analyzed. Results: The evaluation of preliminary phytochemical screening of extracts indicated the presence of carbohydrates, reducing sugars, proteins, amino acids, steroids, flavonoids, terpenoids, saponins, alkaloids, tannins, and phlobatannins. Tuber extracts highlighted effective antibacterial and antifungal activities compared with shoot and flower extracts against all the tested bacteria and fungi. ETOH extract of the tuber observed highest antibacterial activity against Staphylococcus aureus (19 mm), followed by Escherichia coli (18 mm), Micrococcus luteus (17 mm), Pseudomonas aeruginosa (17 mm), and Salmonella abony (16 mm) when compared to shoot and flower extracts. G. superba tuber extracts highlighted effective antifungal activities compared with shoot and flower extracts against all the tested fungi. ETOH extract of the tuber observed highest antifungal activity against Rhizopus oryzae (20.17 mm), followed by Mucor Sp. (19.87 mm), Aspergillus niger (18.02), Candida krusei (17.98 mm), and Candida albicans (16.88 mm) as compared to shoot and flower extracts. Conclusion: Results of the current studies flame lily ETOH extracts showed that the plant has significant antimicrobial activities. The strong antibacterial and antifungal activities of flame lily are due to the presence of tannins and flavonoids present in.


INTRODUCTION
Ayurveda is one of the essential systems of alternative and complementary medicine. Different drug formulations are used in Ayurvedic system of medicines. In addition to other herbal remedies, large portions of its medicines are based on domestic herbs. Complete and advanced knowledge of the medicinal plant is mandatory for everyone working in the Ayurvedic field to find and select the suitable plant for a particular disease [1]. In recent years, interest in medicinal plants has been greatly increased, and the West has taken this issue seriously [2]. The World Health Organization (WHO) presently encourages and promotes traditional medical treatments in national health programs, which are easily available at affordable prices and are culturally accepted. Furthermore, the WHO estimates that approximately one-third of the global population uses herbs and other forms of traditional medicines to treat ailment [3]. Plant-based drugs are safe because there are no or only few side effects [4]. However, the lack of quality control profiles is limited to accepting Ayurvedic drugs. The final product analysis profile has an impact on its performance and safety [5].
In recent years, there has been a huge interest in the use of herbal medicines to cure diseases as there are no side effects compared to synthetic drugs. Flame lily is considered to be the resource of lily colchicines and gloriosine [7]. The flame lily exhibits a broad spectrum of functions to get rid of constipation, anti-inflammatory efficacy, antimicrobial, larvisic test, antibacterial energy, antithrombotic energy, antidepressant energy, enzyme resistance, and serpent bite, skin disease, and respiratory disorders [8][9][10]. Furthermore, it is found suitable for the treatment of injuries and sprains, bitterness, chronic injuries, hemorrhoids, cancers, epadons, nitrinal vaginal emission, leprosy, labor pain, and abortion [11]. It also cures injuries, skin problems, flu, inflammation, piles, blood disorders, lung contractions, general body toner, and poison [12]. Therefore, new drugs expect plant resources for an improved and cheaper alternative from natural products. Treatment standards of these plants are fixed in various phytocompounds by constructing a stable physiological activity in the human body [12]. Various parts of G. superba have different types of applications, in particular, in traditional medical practice. For the treatment of injuries and sprains, bitterness, chronic injuries, hemorrhoids, cancers, epadons, nitrinal JOTHI UCHIMAHALI 1 , ANJELIN JEBAMALAR 1 , GAJALAKSHMI DURAIKANNU 2 , SIVAKUMAR THIRUMAL 3 *

Chemical group tests of the extract
Various chemical groups extracted were performed by phytochemical studies [14]. In each test, 10% (w/v) extraction solution was taken personally to be tested.

Active principle analysis
• Test for steroids and terpenoids: 9 ml ETOH was added to extract and refluxed to for a few minutes. Each filtrate was concentrated in boiling water to 2.5 mL. 5 mL of distilled water was added to a concentrated solution, and the mixture is allowed to stand for 1 h and the wax material is filtered. This filtrate was extracted using the separating funnel using 2.5 mL chloroform. 1 mL of concentrated H 2 SO 4 was added to 0.5 mL chloroform extract in a test tube carefully. A reddish-brown interface showed steroids. • The chloroform extract was evaporated and dehydrated to 0.5 ml on a water bath and heated with 3 ml of concentrated H 2 SO 4 for 10 min on water bath. The presence of gray color showed terpenoids.

Test for flavonoids
About 20 mg of the extract from each of different solvents with 10 ml ETOAC is boiling water for 3 min. The mixture was filtered and the filtrate was used for the following tests:  [17]. The inoculum preparation involved in the growth of fungus on PD agar slants for 1 week at 35°C. The slant was wiped off with a sterile cotton swap and shifted to a sterile tube with fresh PD broth (50 ml). The sterile tubes were then dazed for 5 min and appropriate dilutions were made to estimate the number of cells by microscopic enumeration with a hemocytometer. The final inoculums concentration was maintained to approximately 1.0×10 6 cells/ml. vaginal emission, leprosy, labor pain and abortion [13]. Injuries, skin problems, flu, inflammation, piles, blood disorders, lung contractions, general body toner, poison [11]. The major aim of this work was focused to perform phytochemical and antimicrobial activities on the whole plant extracts of G. superba.

Determining antifungal activity
Each well was filled with plant extracts (100 mL) serially diluted 50% and included by water in 96-well microtiter plates and 100 ml of fungal cultures were added [18]. The amphotericin B was used as an antibiotic reference and 100% acetone was a negative control. The previous concentration of the acetone in the microplate formerly showed [19] that the fungus did not have any influence on fungal growth. A display of growth 40 ml of 0.2 mg/ ml p-iodonitrotetrazolium violet (INT) dissolved in water and filled to the microplate well. The enclosed microplate was incubated for 3-5 days in the plastic bag at 100% relative humidity at 35°C after sealing a plastic bag to minimize fungal contamination in the laboratory. The minimal inhibitory concentration was recorded as the lowest concentration of extract that could prevent the development of antifungal growth. Colorless tetrazolium salt acts as an electron receiver and is inhibited by a red-colored formazan developed with biological active organisms [18]. If the growth of the fungal was prohibited, a well-known solution is clear or a significant reduction in the severity of the color after it is attached to the INT.

Statistical analysis
Experiments have been performed in triplicates and the results have been expressed as mean ± standard deviation. The statistical analysis was made with origin software (OriginPro Evaluation, 2018).

Qualitative phytochemical analysis
The bioactive chemicals examined in the shoots, flowers, and tubers of G. superba whole plant extracts revealed the presence, absence, and not detected of compounds as listed (Tables 1 and 2).

Antibacterial activity
Organic solvents DMSO, ETOAC, and ETOH of tuber extracts displayed more pronounced antibacterial activity than shoot and flower extract. Antibacterial activity extended with the increase inside the attention of the shoot, flower, and tuber extract. The maximum zone of inhibition (19.00±0.45 mm) was exhibited by ETOH tuber extract of G. superba. The DMSO, ETOAC, and ETOH tuber extract confirmed maximum area of inhibition in the various concentrations (25,75, and 125 µL/ml) against shoot and flower as shown in Fig. 1.
This activity can be due to the potential ability of the secondary metabolites to shape a complex with extracellular proteins and with the cell wall of microorganism [29]. The inhibition effect of the methanol leaf extract, on the increase of bacteria, might be due to better solubility of these secondary metabolites, which showed the presence of a wide spectrum of antibiotic compounds [30]. The organic solvent of the plant extract (tuber) renders greater antibacterial ability than the Tannins Aromatic acids ++ +++  shoot and flower extract. This result can be attributed to the polarity of the solvent used for extraction, essential bioactivity, and capability to diffuse in media utilized in assay [31,32]. Therefore, in many elements of the plant for herbal safety against microbial infection are packed with phytochemical substances. The phytochemical analysis revealed the presence of alkaloids, flavonoids, terpenoids, tannins, saponins, aromatic acids, phenolic compounds, xanthoproteins, triterpenoids, amino acids, carbohydrate, reducing sugar, and proteins in shoot, flower, and tuber extracts. Therefore, phytochemical products can be responsible for bacterial activity of G. superba.

Antifungal activity
The antifungal activity of the whole plant shoot, flower, and tuber extracts in different solvents was studied with the aid of agar well diffusion method against five pathogenic fungal species and measured the zone of inhibition for each fungal strain at various concentrations of 25, 75, and 125 µl/ml as shown in Fig. 2 ETOHs solvent extracts of the tuber of G. superba showed highest inhibitory activity against selected fungi than DMSO and ETOAC. The current results confirmed with the previous study, DMSO, ETOAC, and ETOH tuber extract of G. superba which showed potential antifungal activity at 1 mg/disc against to C. albicans, C. krusei, A. niger, R. oryzae, and Mucor sp. [33]. The methanol and ETOH of whole plant extract showed potential antifungal activity against to F. equiseti, B. theobromae, and C. corchori [34]. The phytochemical screening revealed that plant secondary metabolites present in the extracts can agitate the fungal cell wall and also cause the discharge of cellular components such as ions and intracellular proteins which, in turn, stop the progress of the growth of fungi. The plant metabolites saponins, tannins, and alkaloids are known for antimicrobial activity which has been documented well [35]. Many scientists tried to explain the plausible mechanism of action of the antimicrobial activity of plant extracts. Triterpenoids or saponins present in the extracts could create pore-like structures and are responsible for the dissipation of membrane electrical potential or membrane proton motive force, and thus, membrane annihilation occurs [36].

CONCLUSION
Whole plant (shoot, flower, and tuber) extracts showed antibacterial and antifungal activity with maximum inhibition against to selected microorganisms. ETOHs of tuber exhibited maximum antibacterial and antifungal activity. Phytochemical screening revealed that alkaloids, triterpenoids, phenols, saponins, and flavonoids could be responsible for the antimicrobial activities of the G. superba whole plant extracts. It is evident from the current results that compounds of G. superba can be used as antimicrobial agents and ingredients in the human pathogenic diseased formulations in the different pharmaceutical fields.