Introduction

Bacteria or fungi that are associated with plant tissues either within or between the plant cells and those that do not cause any diseases are known as endophytes ¹. Endophytic bacteria have been isolated from different parts of the plant and inhabit most of the plant species ². Bioactive metabolites produced by endophytes have been documented and are found to have antibiotic, antiviral, anticancer, antiinflammatory and antioxidant activities. Compared to soil related microbes, endophytes closely associated with their host plant produce biological molecules greater in number and diversity. Therefore, there is greater possibility to screen unique and active substances from endophytes and to discover new bioactive compounds. Compared to plants, endophytes are sustainable sources of natural products and can be used for large scale production of biologically active compounds to meet the industrial demands as it is easier to scale up the fermentation process of microbes. Although all plants seem to harbour endophytes with some bioactive content and activities, the plants that have medicinal value are of particular interest. There lies a great opportunity to discover distinct and fascinating microorganism among countless plants in various locations and environs. One of the rationales for selecting a plant to isolate endophytes and to discover natural product is to opt plants that have an ethnobotanical history and those that have specific uses or interesting values. Sometimes, the curative abilities of the herbal plants may be attributed to the endophytes of the plant than the natural products of the host plant. Ultimately, plants with ethnobotanical history are reasonable candidates for study, since the therapeutic uses for which the plant may have been chosen relates more to the microflora inhabiting the plant than to the compounds present in plant itself³.

Cissus quadrangularis is a traditional medicinal plant widely used in India and has varied therapeutic claims. The use of the plant to heal fractures and to set bones is stressed in the ayurvedic literature, the Nigathus. The plant possesses noteworthy phytoconstituents that back its several ameliorative activities besides bone remineralization. Today’s pharmacological study over Hadjod proves the ancient classical references of the plant and reestablishes the plant’s potentiality to cure several diseases. The extracts of Cissus possess antioxidant and anticancerous potential⁴.        

The stem of Cissus quadrangularis comprises excellent antioxidants such as vitamin C, carotenoids, calcium and steroids. In ayurvedic and modern drug development areas the plant is treated as a versatile medicinal plant for its worthy therapeutic uses. It does not produce any poisonous effects when consumed ⁵.

Numerous reports are available on diversity of endophytic bacteria and fungi in medicinal plants, but there are meagre reports on endophytic bacteria from Cissus quadrangularis. Endophytic bacteria from Cissus quadrangularis may produce similar compounds as that of the host plant or exhibit bioactivity. Therefore, the present study aims to identify and analyze the bioactive potential of endophytic bacteria isolate CqB14 associated with medicinal plant Cissus quadrangularis.

Material and Methods

Isolation of Endophytic Bacteria and Extraction of Extracellular Compounds

The plant was collected from Department of Plant Biology and Plant Biotechnology, Ethiraj College for Women (Autonomous), Chennai, Tamil Nadu. The plant sample was identified and authenticated at Botanical Survey of India (BSI) Coimbatore, Tamil Nadu (Confirmation I.D No: BSI/SRC/5/23/2018/Tech/1884) as Cissus quadrangularis L. Fresh and healthy stem was surface sterilized by standard methods³. The stem was washed thrice with sterile distilled water. After surface sterilization the outer part of the stem was excised off and the remaining part of the stem was sliced into thin sections. The sections of stem were placed over Nutrient Agar plates and were incubated for 2-4 days. Aseptic conditions were maintained throughout. The endophytic bacteria emerging from the sections of stem were selected and purified by subculturing. Each pure culture was inoculated in Nutrient Broth and incubated for 10 days.  Supernatants were collected after centrifugation. The supernatants were subjected to solvent extraction, using ethyl acetate. The concentrated residue of each extract was regarded as crude sample and stored under refrigeration for further use.

Preliminary Qualitative Phytochemical Screening

The extracellular crude extracts of the endophytic bacteria were analysed for the presence of secondary metabolites, namely carbohydrates, phenols, flavonoids, alkaloids and terpenoids by the standard methods.^{6, 7}

HPTLC Analysis

The High Performance Thin Layer Chromatography (HPTLC) analysis was performed using Hamilton syringe and CAMAG LINOMAT 5 instrument at Anchrom, Mumbai. The extracellular ethyl acetate extract (1:2) of bacteria labelled as CqB14 was subjected to HPTLC fingerprinting and was checked for alkaloids in HPTLC analysis. Increasing volume of the extract was loaded on silica gel 60 F 254 coated TLC plate. Chloroform: Toluene: Ethanol (4:4:1) was used as the mobile phase for HPTLC fingerprinting and Toluene: ethyl acetate: methanol: Ammonia 25 % (30:30:15:1) for alkaloids. Images of the developed TLC plates were taken at white light, UV 254 nm and UV 366 nm in a photo documentation chamber. The developed TLC plates were derivatized with anisaldehyde sulfuric acid reagent and dragendorff’s reagent for fingerprinting and detection of alkaloids respectively. The TLC plates were fixed in scanner stage (CAMAG TLC SCANNER 4) and scanned. The peak table and peak densitogram were noted.

Antioxidant Assay

The antioxidant activity of the extracellular ethyl acetate extract of the endophytic bacteria CqB14 was tested by DPPH assay. The method described by Blois⁸ was used to check the ability of the extracts to reduce the DPPH radical (1, 1-diphenyl-2-picrylhydrazyl). A stock solution of 10 mg/ml concentration was prepared using the extract. Different concentrations of the extract (200, 400, 600, 800, 1000 µg) were added to methanolic solution of DPPH. The reaction mixture was incubated and the absorbance was recorded at 517 nm. The experiment was repeated three times. Ascorbic acid was used as a standard. The annihilation activity of free radicals was calculated in percent inhibition according to the formula Percent Inhibition = (A of control – A of Test) / A of control × 100.

GCMS Analysis

The extracellular extract of CqB14 was subjected to Gas Chromatography-Mass Spectrometry (GC-MS) to predict the compounds present. The Clarus 680 GC used in the analysis, employed a fused silica column, packed with Elite-5MS (5% biphenyl 95% dimethylpolysiloxane, 30 m × 0.25 mm ID × 250μm df) and the components were separated using Helium as carrier gas at a constant flow of 1 ml/min. The injector temperature was set at 260°C during the chromatographic run. One μL of extract sample was injected into the instrument, the oven temperature was as follows: 60 °C (2 min); followed by 300 °C at the rate of 10 °C min^-1, and 300 °C, where it was held for 6 min and total run time 32 min. The mass detector conditions were: transfer line temperature 240 °C; ion source temperature 240 °C; and ionization mode electron impact at 70 eV, a scan time 0.2 sec and scan interval of 0.1 sec. The spectrums of the components were compared with the database of spectrums of known components stored in the GC-MS NIST library.

Effect of the Extract on Saos-2 Cell Line

Osteosarcoma cell line Saos-2 was procured from National Centre for Cell Science (NCCS), Pune, India. The cells were maintained in Minimal Essential Media supplemented with 10 % FBS, penicillin in 5 % CO₂ at 37 ºC. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay was used to assess the inhibitory effect of the extract at various concentrations⁹. Using the microplate reader, the absorbance at 570 nm was recorded to determine the viable cells. The average absorbance values were deduced from the absorbance values observed three times. Percent inhibition =Control – Test/Control X 100

Identification of Endophytic Bacteria

The endophytic bacterium CqB14 was identified by 16S rRNA gene sequence. The genomic DNA of endophytic bacteria was extracted.  Bacterial universal primers were engaged to amplify 16S rDNA in the Polymerase Chain Reaction (PCR) wherein genomic DNA was the template. NCBI BLAST was used to find 16s rRNA genes homologous to the CqB14 sequence. Highly similar sequences were chosen and fed into Mega X. The phylogenetic tree was constituted using the Maximum Likelihood statistical method with bootstrapping as the test of phylogeny. Thousand bootstrap replications were performed to assess nodal support in the tree¹⁰.

Results and Discussion

Preliminary Phytochemical Screening

The preliminary phytochemical analysis of extracellular ethyl acetate extract of the endophyte labelled as CqB14 revealed the presence of carbohydrates, phenols, flavonoids and alkaloids. Similarly, the extract of aerial parts of Cissus quadrangularis showed the presence of carbohydrates, alkaloids, flavonoids, glycosides, saponins, steroids, tannins, triterpenoids ¹¹. The presence of these metabolites indicates that the extract may possess bioactivities.

HPTLC Analysis

HPTLC profiling has been attempted by researchers to substantiate the presence of bioactive compounds ¹². One of the several advantages of HPTLC is the ability to analyze crude samples containing multicomponent. HPTLC application has been proven in analysis of fermentation broth ^13,14. The HPTLC analysis of crude extract of CqB14 showed the presence of peaks before and after derivatization with anisaldehyde. The number of peaks and Rf values differ as per the qualitative variations of the components. The various HPTLC chromatograms are shown in figures 1-10. The chromatograms show separation of constituents without any tailing. Brownish band (RF 0.5) after derivatization with dragendroff’s reagent confirms the presence of alkaloids¹⁵. The Rf values and peak area % are tabulated in table 1 and 2. It is observed in the above HPTLC studies that the extracellular ethyl acetate extract of CqB14 contain a range of chemical constituents with different Rf values including alkaloids. Alkaloids are significant class of chemical compounds that provide a rich reservoir for drug discovery. The antioxidant activities of polyphenols and alkaloids have been demonstrated in several studies. Numerous alkaloids isolated from natural herbs display antiproliferation and antimetastatic effects on various types of cancers both in vitro and in vivo¹⁶. The developed fingerprint analysis will help to isolate and identify new compounds, which will offer a possibility to discover lead molecules for drug development.

Figure 1: HPTLC chromatogram 1a) white light 1b) 254 nm 1c) 366 nm for different volumes of extract Click here to View Figure

Figure 2: HPTLC chromatogram after derivatizing with anisaldehyde 2a) white light 2b) 366 nm for different volumes of extract Click here to View Figure

Figure 3: HPTLC densitometric chromatogram at 254 nm, track 5 Click here to View Figure

Figure 4:  HPTLC densitometric chromatogram at 366 nm, track 5 Click here to View Figure

Figure 5: HPTLC densitometric chromatogram after derivatizing with anisaldehyde at 540 nm, track 5. Click here to View Figure

Figure 6: HPTLC densitometric chromatogram after  derivatizing with anisaldehyde at 366 nm, track 5 Click here to View Figure

Table 1: Rf values and area % of HPTLC chromatogram of extracellular ethyl acetate extract of CqB14, track 5

Figure 7:  HPTLC alkaloid chromatogram 7a) white light 7b) 254 nm 7c) 366 nm for different volumes of extract Click here to View Figure

Figure 8:  HPTLC alkaloid chromatogram after derivatizing with dragendorff Click here to View Figure

Figure 9: HPTLC alkaloid densitometric chromatogram at 254 nm, track 4 Click here to View Figure

Figure 10: HPTLC alkaloid densitometric chromatogram at 366 nm, track 4 Click here to View Figure

Table 2:  Rf values and area % of HPTLC alkaloid profile chromatogram of extracellular ethyl acetate extract of CqB14, track 4

Antioxidant Activity

In present study, extracellular ethyl acetate extract of endophytic bacteria CqB14 was investigated for antioxidant potential by using DPPH method. An increase in antioxidant activity was noted as the concentration of the extracellular extract was increased. The extract showed antioxidant activity of 90.23% at 1000µg/ml [figure 11]. This antioxidant potential may be due to the various metabolites detected by HPTLC. It is reported that 100 ppm concentration of ethyl acetate extracts of Cissus quadrangularis fraction exhibited 61.6% in the 1,1-diphenyl-2-picrylhydrazyl system. This fraction showed the presence of sterols, vitamin C, and tannins as phytoconstituents¹⁷. Antioxidant activity of Cissus quadrangularis extract is also reported by Badami et al¹⁸. The endophytic bacteria may contribute towards the antioxidant activity of the Cissus quadrangularis.

Figure 11: Antioxidant activity by DPPH assay Click here to View Figure

GC-MS analysis

The GC-MS of extracellular crude extract of CqB14 predicted 7 major compounds [Fig 12 and Table 3]. GC-MS analysis indicated that the bacterium CqB14 produced an amino compound 2-Myristynoyl-Glycinamide having antimicrobial activity¹⁹. Benzaldehyde, 4-methoxy was found to be present at a highest percent (peak area 69.5%) among the seven predicted compounds which is reported to be an insect repellant and also used as flavouring ingredient. Benzaldehyde, 4-methoxy also possesses anticancer and antifungal activities²⁰. Acetophenone, 4′-methoxy an aromatic phenol ketone is reported to be a flavouring ingredient and is used as a component of perfumes and as chemical intermediate in the manufacture of pharmaceuticals, resins, flavouring agents²¹. Thus, the results obtained confirm the therapeutic potency of the endophyte CqB14 and forms a good basis for the selection of the isolate for further phytochemical and pharmacological investigation.

Figure 12: GC-MS spectrum of extracellular extracts of CqB14. Click here to View Figure

Table 3: GC-MS analysis of extracellular extracts of CqB14

MTT Assay Using Saos-2 Osteosarcoma Cell Lines

The use of Cissus quadrangularis in the management of bone and joint disorders such as osteoporosis, osteoarthritis, and rheumatoid arthritis has been documented in native medicine. The anti-proliferative properties of the Cissus quadrangularis methanolic extract from aerial parts against MG63 osteosarcoma cells were shown by Suresh et al using cytotoxicity assay²². In this study the anticancer potential of the extracellular crude extract of the endophyte CqB14 was tested using MTT assay against Saos-2 osteosarcoma cell line. MTT assay is a colorimetric assay in which 3-(4,5- dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) enters the cells. In the mitochondria the yellow colored MTT is reduced to an insoluble purple colored formazan product in presence of mitochondrial succinate dehydrogenase. The cells are then solubilized with an organic solvent (e.g. DMSO) and the released, solubilized formazan reagent is measured using spectrophotometer. Since reduction of MTT can only occur in living cells, the level of activity indicates viability of the cells. The generation of reducing equivalents in metabolically active cells forms the basis of MTT assay which helps quantify the viable cells. Greater the number of viable cells higher is the absorbance value²³. Through MTT assay it was observed that as the concentration of the extracellular crude extract of CqB14 was increased the viability of Saos-2 osteosarcoma cells decreased [Figure 13]. The extract may serve as a source of therapy against bone tumours. However detailed study to determine the primary mode of antiosteosarcoma effect needs to be carried out.

Figure 12: GC-MS spectrum of extracellular extracts of CqB14. Click here to View Figure

Identification of Potential Endophytic Bacteria

The endophytic bacteria CqB14 was characterized by 16S rRNA sequencing. BLAST analysis revealed 100% similarity with Pseudomonas azotoformans.  A phylogenetic tree was constructed using MEGA X software and bootstrap algorithm [Fig. 14]. The internal node connecting the query sequence CqB14 with Pseudomonas azotoformans, Pseudomonas libianensis, Pseudomonas reactans, Pseudomonas fluorescens and Pseudomonas gessardii with a bootstrap value of 66% was obtained. The sequence was deposited at NCBI genbank and accession number MN559400 was obtained. Mostly, endophytic bacteria isolated from medicinal plants belongs to Pseudomonas and Bacillus species²⁴. To the best of our knowledge this is the foremost study which reports the isolation of endophytic bacteria similar to Pseudomonas species from Cissus quadrangularis.

Figure 14: Phylogeny tree constructed using Mega X Click here to View Figure

Conclusion

Bacterial endophytes have several capabilities that it can be utilized in pharmaceutical and drug discovery. Endophytes related with plants having medicinal significance serve as a potential source of natural products and for developing new agents exhibiting a wide range of bioactivities. A single endophyte may yield a variety of bioactive metabolites. The utilization of highly automated HPTLC, can be considered as a useful tool in the analysis of complex mixtures of natural products. In the present investigation the endophytic bacteria CqB14 isolated from ethnomedicinal plant Cissus quadrangularis seems to be an alternative source of natural antioxidant and anticancer compounds. CqB14 identified to be similar to Pseudomonas species is capable of producing various secondary metabolites admissible as therapeutics against human ailments and clinically effective bioactive compounds. So, it could be proposed as an organism of pharmaceutical interest. Further studies will need to be undertaken by isolating individual secondary metabolite and determining the biological activities of individual constituents which may hopefully yield good results.

Acknowledgement

The authors thank former and present Head, the Faculty members, supporting staff of the Department of Plant Biology and Plant Biotechnology and the Principal, Ethiraj College for Women, (autonomous) Chennai – 600 008, for their valuable support and encouragement throughout the entire period of research. We would also like to express our thanks for the facilities extended by the Central Instrumentation Centre of Ethiraj College for Women.

Funding Source

No funding sources.

Conflict of Interest

Authors declare no conflict of interest.

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