{"id":53541,"date":"2023-12-31T11:24:11","date_gmt":"2023-12-31T11:24:11","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=53541"},"modified":"2024-01-05T06:20:17","modified_gmt":"2024-01-05T06:20:17","slug":"in-vitro-assessment-of-antioxidant-and-antimicrobial-activity-of-field-grown-and-tissue-cultured-fruit-callus-of-nothapodytes-nimmoniana","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no4\/in-vitro-assessment-of-antioxidant-and-antimicrobial-activity-of-field-grown-and-tissue-cultured-fruit-callus-of-nothapodytes-nimmoniana\/","title":{"rendered":"In-Vitro Assessment of Antioxidant and Antimicrobial Activity of Field Grown and Tissue Cultured Fruit Callus of Nothapodytes nimmoniana"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Plant parts and plant-derived drugs have been\nused in the health care system since ancient human civilization.1<\/sup>\nNatural antioxidants are mostly found in plants, which also create a variety of\nsecondary metabolites with antioxidant properties and potential medicinal\napplications. Several complicated illnesses are prevented and treated with\nmedication formulations based on antioxidants.2<\/sup> Due to oxidative\nstress, free radicals are produced, and vital vitamins and enzymes are\ndepleted, leading to oxidative illnesses. Examples of free radicals include the\nhydroxyl radical, the nitric oxide radical, singlet oxygen, hydrogen peroxide,\nthe superoxide anion radical, the hypochlorite radical, and other lipid\nperoxides. All these free radicals have the potential to interact with small\nmolecules such as biological membranes, nucleic acids, proteins, enzymes, and\nother small molecules to cause cellular damage. Also, these free radicals play\na role in a variety of medical conditions, including diabetes, cancer,\nneurological illnesses, and inflammatory diseases.3<\/sup><\/p>\n\n\n\n

Antioxidants protect the human system by\ncooperatively and synergistically scavenging free radicals. Vitamins,\nalkaloids, phenolic acids, flavonoids, terpenoids, tannins, lignins, stilbenes,\ncoumarins, quinones, betalains, amines, and other metabolites with high\nantioxidant activity are only a few of the free radical-scavenging compounds\nfound in plants.4<\/sup> Therefore, intensive research is being carried out\non natural antioxidants.<\/p>\n\n\n\n

Natural products are still one of the main\nsources of new medication compounds today. They are derived from a wide range\nof animal species, plants, eukaryotic microbes, prokaryotic bacteria, and\nplants. The majority of antibacterial compounds that have been found so far\ncome from microbial and plant sources. Because of this, finding new antibiotics\nis a particularly crucial goal.5<\/sup> In vitro testing of extracts and\npure medicines as possible antimicrobial agents has been done for antibacterial\nactivity.<\/p>\n\n\n\n

One of the most effective cancer treatments of\nthe twenty-first century is camptothecin (CPT), a monoterpene indole alkaloid.6<\/sup>\nDNA topoisomerase I is the biological target of camptothecin, and various\nanalogues have been produced as possible therapeutics. The complete cure of\nlung, breast, uterine, and cervical cancers has been demonstrated to be\npossible with CPT.7<\/sup> Currently, topotecan and irinotecan, two\nwater-soluble camptothecin compounds, have been used to treat ovarian and\ncolorectal cancer, respectively.8<\/sup> Camptotheca acuminata, a Chinese\ndeciduous tree, is where CPT was initially found. Nothapodytes nimmoniana<\/em>\n(N. nimmoniana<\/em>) was discovered to have the highest concentration of CPT\n(0.3% w\/w).9<\/sup><\/p>\n\n\n\n

A little tree known as N. nimmoniana<\/em> (J.\nGraham) Mabberly (Family Icacinaceae), formerly known as Nothapodytes\nfoetida<\/em> Sleumer and Mappia foetida Miers, is found in numerous areas of the\nWestern Ghats of India, certain areas of Assam, the Himalayan foothills,\nCeylon, Burma, and Thailand.10<\/sup> When synthetic sources are not\npresent, the exploitation of naturally occurring populations of N.\nnimmoniana<\/em> from the Western Ghats, India, supplies the world’s demand for\nCPT (US$4045 million in 2002). As a consequence, the species population in the\nWestern Ghats has decreased by almost 20% just in the past ten years.11, 12<\/sup><\/p>\n\n\n\n

Therefore, in the present study, we have\nprepared extracts from different plant parts like leaves, stem, stem bark,\nroots, and fruit, as well as in vitro parts like callus culture and suspension\nculture extracts of N. nimmoniana <\/em>and evaluated them as antioxidants and\nantimicrobials.<\/p>\n\n\n\n

Material and methods<\/strong><\/p>\n\n\n\n

Drugs and\nChemicals<\/strong><\/p>\n\n\n\n

The chemicals used in the\nexperiments were of analytical grade. Ascorbic acid, potassium ferricyanide,\nand ferric chloride were procured from Sigma Chemicals, Mumbai, India. DPPH was\npurchased from Hi Media Chemicals Ltd., Mumbai, India. Nutrient Agar Media, Nutrient\nAgar Broth, SDA and DMSO.<\/p>\n\n\n\n

Collection of plant material<\/strong><\/p>\n\n\n\n

Plant material of Nothapodytes nimmoniana <\/em>was collected from\nMahabaleshwar, Pune (Maharashtra), in the month of February. Different plant\nparts like fruits, leaves, stems, roots, and stem bark were collected for this\nstudy. The plant parts (leaves and fruits) were identified by the National\nInstitute of Science Communication and Information Resources, New Delhi, India.\nNISCAIR\/RHMD\/Consult\/2021\/3823-24-2.<\/p>\n\n\n\n

Extraction\nof natural and in-vitro <\/em>grown plant material:<\/strong><\/p>\n\n\n\n

The Nothopodytes nimmoniana<\/em> plant material was collected, cleaned well, divided\ninto distinct components (roots, leaves, stems, and fruits), and dried at 55\u00baC\nin an air dryer for 48 hours. Separate dried plant pieces were ground into\npowder using a mortar and pestle until they could pass through a 20-mesh filter\nand then packaged in plastic bags. In a conical flask, 2 g of the powdered\nsubstance were combined with 50 ml of methanol and kept in a probe sonicator\n(Labman Pro-250) for the extraction of biomarker compounds. The sonication\nprocess was maintained at three variable conditions: 40-50\u2070C, 05:05 pulse for\n15 min; 40-50\u2070C, 02:02 pulse for 20 min; and 40- 50\u2070C, 07:07 pulse for 10 min.\nVacuum-dried extract was then diluted in methanol to create a variety of\ndilutions.<\/p>\n\n\n\n

Quantitative assessment of\nphytoconstituents<\/strong><\/em><\/p>\n\n\n\n

Determination of total phenolic content<\/strong><\/p>\n\n\n\n

Total phenolic\nlevels were determined using the colorimetric method13<\/sup> and Tannic acid as standard, with a\nslight modification of the tannic acid concentration diluted into several\nconcentrations of 10-100\u03bcg\/ml. 1 mL of extract or each standard solution was\nadded 1.5 ml of Folin-Ciocalteu reagent, and after 5 minutes added 1.5 mL of\n75% NA2CO3 and distilled water up to 10 mL were added. The mixture was\nincubated for 30 minutes in dark conditions at room temperature. After\nincubation, the absorbance was measured using a UV-Vis spectrophotometer at 765\nnm. The total phenolics of methanolic extract were calculated using the Tannic\nacid calibration curve. The results were expressed as Tannic acid equivalent\n(GAE) mg\/g extract.<\/p>\n\n\n\n

Determinations of flavonoid content<\/strong><\/p>\n\n\n\n

Total flavonoid\nlevels determined by the colorimetric method13<\/sup>\nusing quercetin as a standard refer to the procedure that was modified for its\nquercetin concentration. standard quercetin dissolved with methanol diluted to\nconcentrations of 25, 50, 75, and 100 \u03bcg\/ml. 1 ml of each concentration of\nquercetin standard solution and extract, mixed with 4 mL of distilled water and\n0.3 ml of 5% Na2<\/sub>NO3. <\/sub>After 5 minutes, 0.3 ml of 10% AlCl3 <\/sub>was\nadded, and after 6 minutes of incubation, the samples were2 ml of NaOH (1 M)\nwas also added to make up 10 ml vol. by volumetric flask by 2.4 ml distilled\nwater. After incubation, the absorbance was measured using a UV-Vis\nspectrophotometer at 510 nm. Total flavonoids of the methanolic extract were\ncalculated using the quercetin calibration curve. The results were expressed as\nquercetin equivalent (QE) mg\/g extract.<\/p>\n\n\n\n

Antioxidant\nstudies<\/strong><\/p>\n\n\n\n

Determination of\n1, 1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity<\/strong><\/p>\n\n\n\n

Based on the scavenging of stable DPPH free\nradicals, the antioxidant activity was determined.13<\/sup> Various\nconcentrations (100\u2013500 \u03bcg\/mL) of methanolic extracts were added to 1 mL of a\n0.004% methanol solution of DPPH. 2 ml of methanol was added to give a final\nworking volume. The blank sample consisted of 2ml ml of methanol, while the\ncontrol consisted of 2 ml of methanolic DPPH only. The absorbance of samples,\nblank, and control was measured at 517 nm using a UV-Vis spectrophotometer\n(Labman). The mixed solution was incubated at room temperature in the dark for\n30 minutes. The percent of DPPH scavenging activity was measured as<\/p>\n\n\n\n

Scavenging activity (%) = (Absorbance of control\nsample – Absorbance of test sample) \/ (Absorbance of control) x 100<\/p>\n\n\n\n

Determination of\nreducing power<\/strong><\/p>\n\n\n\n

From the stock solution, sample solutions\ncomprising 10\u2013100 g extracts were created. A 1 mL aliquot of the extract was\ncombined with 1 mL of 1% (w\/v) potassium ferricyanide and 1 mL of 0.2 M\nphosphate buffer with a pH of 6.6. The mixture was incubated at 50\u00b0C for 20\nminutes. The mixture was centrifuged at 3000 rpm for 10 minutes after adding 1\nmL of 10% w\/v trichloroacetic acid. 1 mL of the supernatant was mixed with 0.2\nmL of a 0.1% (w\/v) ferric chloride (FeCl3<\/sub>)\nsolution and 1 mL of distilled water. A UV-Vis spectrophotometer was used to\nmeasure the absorbance at 700 nm. The reducing power of ascorbic acid as a\nstandard was also determined. The results were expressed as absorbance values.\nThe increased absorbance of the reaction mixture indicates an increase in\nreducing power.14<\/sup><\/p>\n\n\n\n

Determination of H2<\/sub>O2\n<\/sub>radical scavenging activity<\/strong><\/p>\n\n\n\n

The extract’s\ncapacity to scavenge H2<\/sub>O2<\/sub> was established. H2<\/sub>O2<\/sub>\nsolution (0.6 mL, 40 mM) was added to 100 \u03bcl (0.1 ml) of extract samples or\nstandards (10\u2013100 \u03bcg\/mL). A solution of H2<\/sub>O2 <\/sub>was prepared\nin 50 mM PBS (pH 7.4) and diluted with 4 ml PB. The H2<\/sub>O2 <\/sub>concentration\nwas determined spectrophotometrically at 230 nm. The absorbance was measured\nafter 10 minutes against a blank (PBS without H2<\/sub>O2<\/sub>)and\na control (PBS with H2<\/sub>O2<\/sub>).15<\/sup> The antioxidant\nactivity of the extracts was expressed as IC50.<\/strong><\/p>\n\n\n\n

Antimicrobial activity\n16<\/sup><\/strong><\/p>\n\n\n\n

Antibacterial\nactivity of the plant extracts<\/strong><\/p>\n\n\n\n

Well\ndiffusion technique<\/strong><\/p>\n\n\n\n

Preparation of\ntest compound <\/strong><\/p>\n\n\n\n

The test extracts were evaluated for their\nantimicrobial properties, and amoxicillin was used as a standard test\nsubstance. During 24 hours at 37\u00b0C, all four pathogens were cultured in\nnutrient broth. Leaves, roots, stems, stem bark, fruits, and in vitro-grown\ncallus and suspension culture extracts were prepared in DMSO solutions. The\nDrug concentration was prepared as 100mg\/ml for the antimicrobial test.<\/p>\n\n\n\n

Bacterial\ninoculum preparation<\/strong><\/p>\n\n\n\n

The antibacterial potency of each plant\nextract was evaluated using four bacterial strains: Escherichia coli<\/em> (MTCC 40), Staphylococcus\naureus <\/em>(MTCC 740), Klebsiella\npneumoniae<\/em> (MTCC 109), and Pseudomonas\naeruginosa<\/em> (MTCC 741). The bacterial strains were provided by MTCC,\nthe institute of microbial technology (IMTECH), Chandigarh. On Nutrient agar\nslants, each bacterial strain was subcultured for an entire night at 35\u201337\u00b0C.\nThe bacterial inoculum was collected using 5 ml of sterile saline water,\ndisciplined for absorbance at 580 \u00b5m using a spectrophotometer, and diluted to\nachieve a viable cell count of 107 CFU\/ml.<\/p>\n\n\n\n

Evaluation of\nantimicrobial activity<\/strong><\/p>\n\n\n\n

The nutrient agar,\nabout 20 mL, was melted, cooled, and then placed into sterile petri plates.\n100\u03bcl of all four pathogens were swabbed on Nutrient agar plates. In every agar\nplate, a 5-mm well was made. The well was filled with 100\u03bcg\/ml test compound\nand Amoxicillin (0.1 mg\/ml), and it was then incubated at 37o<\/sup>C for\n24-48 hours. The zone of inhibition was observed surrounding the wells on the\nplates. Using the well diffusion technique, the most potent plant extracts were\nidentified with high antibacterial activity at 10 mg\/ml.<\/p>\n\n\n\n

Determination of\nminimum inhibitory concentrations (MIC) and <\/strong>Minimum\nbactericidal concentration (MBC)<\/strong><\/strong><\/p>\n\n\n\n

The MIC is defined\nas the minimum antimicrobial drug concentration that suppresses microbiological\ngrowth after 24 hours of incubation. The minimum concentrations of plant\nextract that showed invisible growth (from the inhibition zone of MIC plates)\nwere collected as streaks, and they were then subcultured into nutrient agar\nplates. The plates were checked for bacterial growth corresponding to plant\nextract concentration after 24 hours of incubation at 35 \u00b0C. The minimum\nbactericidal concentration (MBC) was defined as the plant extract concentration\nthat did not show any bacterial growth.<\/p>\n\n\n\n

Antifungal activity<\/strong><\/p>\n\n\n\n

Well\ndiffusion technique<\/strong><\/p>\n\n\n\n

Preparation of\ntest compound <\/strong><\/p>\n\n\n\n

As fluconazole has the capacity to stop\nthe growth of all infections, it was chosen as the standard test substance. A\nfungal strain of Candida Albicans<\/em>\nwas grown in Sabouraud dextrose broth at 28-30\u00baC for 3-5 days. The leaves,\nroot, stem, stem bark, fruits, and in vitro-grown callus and suspension culture\nextracts were taken and prepared in DMSO solution. The Drug concentration was\nprepared at 100mg\/ml for antifungal testing.<\/p>\n\n\n\n

Fungal\ninoculum preparation<\/strong><\/p>\n\n\n\n

The antifungal potency was evaluated using\nthe fungal strain Candida Albicans <\/em>(MTCC\n183). The fungal strain was provided by the Institute of Microbial Technology\n(IMTECH), Chandigarh. A fungal strain was subcultured overnight at 28-30\u00baC for\n3-5 days on Sabouraud Dextrose agar plates.<\/p>\n\n\n\n

Evaluation of\nantifungal activity<\/strong><\/p>\n\n\n\n

In sterile petri dishes, 20 mL of molten\nand cooled Sabouraud dextrose agar medium was added. On agar plates, a loopy\nfungus strain was swabbed. In every agar plate, a 5-mm well was made.\nFluconazole and 100 g\/ml test compounds were added to the well and incubated\nthere for 3-5 days at 28\u201330 \u00baC. The plates were observed for an inhibitory zone\nsurrounding the wells on the plates. Using the well diffusion technique, the\nmost potent plant extracts that demonstrated significant antifungal activity at\n100 mg\/ml were identified.<\/p>\n\n\n\n

Determination of\nminimum inhibitory concentrations (MIC) and <\/strong>Minimum\nfungicidal concentration (MFC)<\/strong> <\/strong><\/strong><\/p>\n\n\n\n

MIC is defined as\nthe minimum concentration of an antifungal dose that suppresses microbial\ngrowth after 3-5 days of incubation. Swabbed fungal inoculum was subcultured\nonto Sabouraud Dextrose agar plates using the lowest concentrations of plant\nextract, which established invisible growth (from the inhibition zone of MIC\nplates). For 3-5 days, the plates were incubated at 28\u201330 \u00b0C, then checked for\nfungus development. The minimum fungicidal concentration (MFC) was chosen as\nthe concentration of plant extract on the freshly infected agar plates that did\nnot show any fungal growth.<\/p>\n\n\n\n

Results and <\/strong>Discussion<\/strong> <\/p>\n\n\n\n

Determination of\nPhyto-constitute content:<\/strong><\/p>\n\n\n\n

Total Phenolic Content (TPC)<\/strong><\/p>\n\n\n\n

Tannic acid\ncalibration curves as standard phenol compounds are presented in Figure. From\nthe results of the Tannic acid calibration curve obtained a regression equation\ny = 0.0021x – 0.0331 with R\u00b2 = 0.9336. The curves with a value of R2 close to\none are linear.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Calibration Curve of Standard (Tannic acid).<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The total phenolic\ncontent of plant extracts and tissue culture extracts was determined as TAE\/g\nextract. The callus and suspension cultures showed the maximum phenolic content\nof 0.89mg\/g and 0.93mg\/g, respectively. Among plant parts, stem bark extract\nand fruit extract showed the maximum phenolic content of 0.52mg\/ml and 0.68\nmg\/ml, respectively. Summarised in Table no. 1.<\/p>\n\n\n\n

Table 1: Total phenolic content in different samples of N. nimmoniana.<\/em><\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Samples<\/strong><\/p>\n<\/td>\n

\n

Total Phenolic Content (TPC) mg\/g<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Leaves<\/p>\n<\/td>\n

\n

0.22<\/p>\n<\/td>\n<\/tr>\n

\n

Roots<\/p>\n<\/td>\n

\n

0.32<\/p>\n<\/td>\n<\/tr>\n

\n

Stem<\/p>\n<\/td>\n

\n

0.47<\/p>\n<\/td>\n<\/tr>\n

\n

Stem Bark<\/p>\n<\/td>\n

\n

0.52<\/p>\n<\/td>\n<\/tr>\n

\n

Fruits<\/p>\n<\/td>\n

\n

0.68<\/p>\n<\/td>\n<\/tr>\n

\n

Callus<\/p>\n<\/td>\n

\n

0.89<\/p>\n<\/td>\n<\/tr>\n

\n

Suspension culture<\/p>\n<\/td>\n

\n

0.93<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Total Flavonoid content estimation<\/strong><\/p>\n\n\n\n

Quercetin calibration curves as standard phenol compounds are\npresented in Figure. From the results of the Quercetin calibration curve\nobtained a regression equation y = 0.013x + 0.0026 with R\u00b2 = 0.9931.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2: Calibration Curve of Standard (Quercetin)<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The total Flavonoid\ncontent of plant extracts and in vitro culture extracts was determined as QE\/g\nextract. The callus and suspension cultures showed maximum flavonoid content of\n0.76mg\/g and 0.82 mg\/g, respectively. Among the plant parts, roots extract and\nfruits extract have maximum phenolic content of 0.24mg\/ml and 0.35 mg\/ml,\nrespectively.<\/p>\n\n\n\n

Table 2: Total Flavonoid content in different samples of N. nimmoniana<\/em><\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Samples<\/strong><\/p>\n<\/td>\n

\n

Total Flavonoid Content (TFC)<\/strong><\/p>\n

mg\/g<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Leaves<\/strong><\/p>\n<\/td>\n

\n

0.09<\/p>\n<\/td>\n<\/tr>\n

\n

Stem<\/strong><\/p>\n<\/td>\n

\n

 0.11<\/p>\n<\/td>\n<\/tr>\n

\n

Stem bark<\/strong><\/p>\n<\/td>\n

\n

0.16<\/p>\n<\/td>\n<\/tr>\n

\n

Roots<\/strong><\/p>\n<\/td>\n

\n

0.24<\/p>\n<\/td>\n<\/tr>\n

\n

Fruits<\/strong><\/p>\n<\/td>\n

\n

0.35<\/p>\n<\/td>\n<\/tr>\n

\n

Callus<\/strong><\/p>\n<\/td>\n

\n

0.76<\/p>\n<\/td>\n<\/tr>\n

\n

Suspension Culture<\/strong><\/p>\n<\/td>\n

\n

0.82<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Antioxidant activity <\/strong><\/p>\n\n\n\n

Determination of DPPH radical scavenging activity<\/strong><\/p>\n\n\n\n

In the DPPH method, the absorbance was measured at 517 nm. A lower\nabsorbance of the reaction mixture indicates higher free radical scavenging\nactivity. Measuring the decrease in absorbance at 517 nm allows for a\nquantitative estimation of the reduction of DPPH radicals, widely used to\nevaluate the free radical scavenging effect of natural antioxidants. 17<\/sup>\nThe results of absorbance and % inhibition showed a decrease in concentration\nof DPPH radical due to the scavenging ability of the extract and standard\nascorbic acid as a reference compound. The antioxidant capacity of N.\nnimmoniana<\/em> is summarised in Table 3. The greatest capacity to scavenge the\nDPPH radical was confirmed for the extracts of suspension culture and fruit\ncallus. It was significantly lowered in suspension culture (35.04%, 42.7%,\n53.48%, 66.77%, 79.55%), and fruit callus extract (38.11%, 44.16%, 51.94%,\n62.07%) at different concentrations. The IC50 values of suspension culture and\nfruit callus culture exhibited significant values of 43.09 mg\/ml and 42.58\nmg\/ml, respectively. A hydrogen ion or an electron may be required for the\nstable free radical DPPH to change into a constant diamagnetic molecule. The\nmethanolic solution of DPPH displays a strong absorbance at 517nm due to odd\nelectrons.<\/p>\n\n\n\n

Table 3: DPPH radical scavenging activity of extract of N. nimmoniana<\/em>.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Samples<\/strong><\/p>\n<\/td>\n

\n

Concentration (\u00b5g\/ml)) and % Inhibition<\/strong><\/p>\n<\/td>\n

\n

IC50 (\u00b5g\/ml)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

10<\/strong><\/p>\n<\/td>\n

\n

20<\/strong><\/p>\n<\/td>\n

\n

30<\/strong><\/p>\n<\/td>\n

\n

40<\/strong><\/p>\n<\/td>\n

\n

50<\/strong><\/p>\n<\/td>\n

\n<\/td>\n<\/tr>\n
\n

Std. Ascorbic acid<\/p>\n<\/td>\n

\n

51.06\u00b10.03**<\/p>\n<\/td>\n

\n

64.78\u00b10.03**<\/p>\n<\/td>\n

\n

72.01\u00b10.02**<\/p>\n<\/td>\n

\n

89.54\u00b10.02**<\/p>\n<\/td>\n

\n

91.57\u00b10.01**<\/p>\n<\/td>\n

\n

40.02<\/p>\n<\/td>\n<\/tr>\n

\n

Leaves<\/p>\n<\/td>\n

\n

15.84\u00b10.01<\/p>\n<\/td>\n

\n

18.14\u00b10.03<\/p>\n<\/td>\n

\n

20.36\u00b10.04<\/p>\n<\/td>\n

\n

22.8\u00b10.01<\/p>\n<\/td>\n

\n

24.27\u00b10.02<\/p>\n<\/td>\n

\n

47.52<\/p>\n<\/td>\n<\/tr>\n

\n

Stem<\/p>\n<\/td>\n

\n

13.34\u00b10.01<\/p>\n<\/td>\n

\n

15.94\u00b10.02<\/p>\n<\/td>\n

\n

17.55\u00b10.01<\/p>\n<\/td>\n

\n

20.3\u00b10.02<\/p>\n<\/td>\n

\n

22.56\u00b10.03<\/p>\n<\/td>\n

\n

48.09<\/p>\n<\/td>\n<\/tr>\n

\n

Stem bark<\/p>\n<\/td>\n

\n

8.01\u00b10.01<\/p>\n<\/td>\n

\n

12.07\u00b10.03<\/p>\n<\/td>\n

\n

14.41\u00b10.02<\/p>\n<\/td>\n

\n

16.57\u00b10.03<\/p>\n<\/td>\n

\n

18.62\u00b10.02<\/p>\n<\/td>\n

\n

47.52<\/p>\n<\/td>\n<\/tr>\n

\n

Roots<\/p>\n<\/td>\n

\n

18.29\u00b10.02<\/p>\n<\/td>\n

\n

20.19\u00b10.04<\/p>\n<\/td>\n

\n

23.98\u00b10.01<\/p>\n<\/td>\n

\n

24.27\u00b10.03<\/p>\n<\/td>\n

\n

26.97\u00b10.02<\/p>\n<\/td>\n

\n

48.39<\/p>\n<\/td>\n<\/tr>\n

\n

Fruits<\/p>\n<\/td>\n

\n

22.94\u00b10.02<\/p>\n<\/td>\n

\n

25.28\u00b10.01<\/p>\n<\/td>\n

\n

28.32\u00b10.02<\/p>\n<\/td>\n

\n

29.96\u00b10.03<\/p>\n<\/td>\n

\n

30.72\u00b10.04<\/p>\n<\/td>\n

\n

45.44<\/p>\n<\/td>\n<\/tr>\n

\n

Fruit callus<\/p>\n<\/td>\n

\n

27.68\u00b10.09<\/p>\n<\/td>\n

\n

38.11\u00b10.07*<\/p>\n<\/td>\n

\n

44.16\u00b10.05*<\/p>\n<\/td>\n

\n

51.94\u00b10.03**<\/p>\n<\/td>\n

\n

62.07\u00b10.04**<\/p>\n<\/td>\n

\n

43.09**<\/p>\n<\/td>\n<\/tr>\n

\n

Suspension Culture<\/p>\n<\/td>\n

\n

35.04\u00b10.04*<\/p>\n<\/td>\n

\n

42.7\u00b10.01*<\/p>\n<\/td>\n

\n

53.48\u00b10.08**<\/p>\n<\/td>\n

\n

66.77<\/strong>\u00b10.03**<\/p>\n<\/td>\n

\n

79.55\u00b10.02 **<\/p>\n<\/td>\n

\n

42.58**<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are given as mean \u00b1 S.D. ** Significant at p\u02c20.01, * Significant at p\u02c20.05. p-value was calculated by comparing with control by ANOVA followed Dunnett\u2019s test, values are expressed as \u00b1 SEM.<\/p>\n\n\n\n
\"\"<\/td>\n\n

Figure 3a: <\/strong>DPPH scavenging activity of <\/strong>extracts o<\/strong>f<\/strong> N. nimmoniana.<\/em><\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 3b: DPPH Scavenging activity and their IC 50 values<\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Determination of\nreducing power<\/strong><\/p>\n\n\n\n

A molecule’s potential\nantioxidant action may be strongly suggested by the reducing power of the\nchemical. When the Fe3+<\/sup>\nferricyanide complex was reduced to the ferrous form (Fe2+<\/sup>) during the reducing power assay,\nreductants (antioxidants) were present in the tested samples. The content of N. nimmoniana<\/em> extracts in the reaction\nmixture was measured by measuring the absorbance at 740 nm, and this linearly\nincreased. The absorbance gained with increasing concentration for both the\nstandard ascorbic acid and sample extracts in the reducing power experiment.\nThe reducing power was shown by absorbance in a concentration dependent manner.\nThe suspension culture and fruit callus culture exhibited high reducing powers\nof 0.278<\/em>, 0.392<\/em>, 0.269, and<\/em> 0.320<\/em> in 75\u00b5g\/ml and 100 \u00b5g\/ml\nconcentrations, respectively. summarised in Table no. 4.<\/p>\n\n\n\n

Table 4: Reducing power activity of methanolic extract of N. nimmoniana<\/em>.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Extracts<\/strong><\/p>\n<\/td>\n

\n

Concentration (\u00b5g\/ml) and Absorbance <\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

10<\/strong><\/p>\n<\/td>\n

\n

25<\/strong><\/p>\n<\/td>\n

\n

50<\/strong><\/p>\n<\/td>\n

\n

75<\/strong><\/p>\n<\/td>\n

\n

100<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Std. Ascorbic acid<\/p>\n<\/td>\n

\n

0.097\u00b10.01<\/p>\n<\/td>\n

\n

0.137\u00b10.03<\/p>\n<\/td>\n

\n

0.243\u00b10.07<\/p>\n<\/td>\n

\n

0.349\u00b10.04<\/p>\n<\/td>\n

\n

0.471\u00b10.02<\/p>\n<\/td>\n<\/tr>\n

\n

Leaves<\/p>\n<\/td>\n

\n

0.06\u00b10.07<\/p>\n<\/td>\n

\n

0.108\u00b10.01<\/p>\n<\/td>\n

\n

0.116\u00b10.02<\/p>\n<\/td>\n

\n

0.254\u00b10.02<\/p>\n<\/td>\n

\n

0.259\u00b10.03<\/p>\n<\/td>\n<\/tr>\n

\n

stem<\/p>\n<\/td>\n

\n

0.022\u00b10.03<\/p>\n<\/td>\n

\n

0.103\u00b10.06<\/p>\n<\/td>\n

\n

0.165\u00b10.05<\/p>\n<\/td>\n

\n

0.213\u00b10.01<\/p>\n<\/td>\n

\n

0.262\u00b10.07<\/p>\n<\/td>\n<\/tr>\n

\n

Stem bark<\/p>\n<\/td>\n

\n

0.025\u00b10.02<\/p>\n<\/td>\n

\n

0.094\u00b10.04<\/p>\n<\/td>\n

\n

0.162\u00b10.01<\/p>\n<\/td>\n

\n

0.202\u00b10.02<\/p>\n<\/td>\n

\n

0.244\u00b10.01<\/p>\n<\/td>\n<\/tr>\n

\n

Roots<\/p>\n<\/td>\n

\n

0.035\u00b10.05<\/p>\n<\/td>\n

\n

0.103\u00b10.04<\/p>\n<\/td>\n

\n

0.221\u00b10.01<\/p>\n<\/td>\n

\n

0.262\u00b10.02<\/p>\n<\/td>\n

\n

0.263\u00b10.00<\/p>\n<\/td>\n<\/tr>\n

\n

Fruits<\/p>\n<\/td>\n

\n

0.038\u00b10.01<\/p>\n<\/td>\n

\n

0.065\u00b10.03<\/p>\n<\/td>\n

\n

0.126\u00b10.06<\/p>\n<\/td>\n

\n

0.134\u00b10.04<\/p>\n<\/td>\n

\n

0.243\u00b10.05<\/p>\n<\/td>\n<\/tr>\n

\n

Fruit callus<\/p>\n<\/td>\n

\n

0.067\u00b10.02<\/p>\n<\/td>\n

\n

0.093\u00b10.07<\/p>\n<\/td>\n

\n

0.126\u00b10.03<\/p>\n<\/td>\n

\n

0.269\u00b10.01*<\/p>\n<\/td>\n

\n

0.320\u00b10.04**<\/p>\n<\/td>\n<\/tr>\n

\n

Suspension Culture<\/p>\n<\/td>\n

\n

0.071\u00b10.04<\/p>\n<\/td>\n

\n

0.117\u00b10.07<\/p>\n<\/td>\n

\n

0.134\u00b10.01<\/p>\n<\/td>\n

\n

0.278\u00b10.02*<\/p>\n<\/td>\n

\n

0.392\u00b10.01**<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are given as mean \u00b1 S.D. (n=3) ** Significant at p\u02c20.01, * Significant at p\u02c20.05. p-value was calculated by comparing with control by ANOVA followed Dunnett\u2019s test, values are expressed as \u00b1 SEM.<\/p>\n\n\n\n
\"\"<\/td>\n\n

Figure 4: <\/strong>Reducing potential of standard and methanolic <\/strong>o<\/strong>f<\/strong> N. nimmoniana<\/em><\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Determination of H2<\/sub>O2\n<\/sub>radical scavenging activity<\/strong><\/p>\n\n\n\n

The H2<\/sub>O2 <\/sub>radical\nitself is not extremely reactive, but it can occasionally be hazardous to cells\nbecause it causes the cell to produce the OH radical. The H2<\/sub>O2<\/sub>\nradical was considerably scavenged by N.\nnimmoniana<\/em> extracts in a concentration-dependent manner. The\npercentage inhibition of suspension culture and fruit callus culture extract\nsignificantly exhibited scavenging activity, respectively (42.27%, 50.14%,\n60.5%), (43.18%, 58.76), as summarised in Table 5. The IC50 values of\nsuspension culture and fruit callus culture extract both significantly\nexhibited 41.81 \u00b5g\/ml and 43.19 \u00b5g\/ml respectively.<\/strong><\/p>\n\n\n\n

Table 5: H2<\/sub>O2 <\/sub>  radical scavenging activity of methanolic extract of N. nimmoniana<\/em>.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Extracts<\/strong><\/p>\n<\/td>\n

\n

Concentration (\u00b5g\/ml) and % Inhibition<\/strong><\/p>\n<\/td>\n

\n

IC 50 (\u00b5g\/ml)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

10<\/strong><\/p>\n<\/td>\n

\n

25<\/strong><\/p>\n<\/td>\n

\n

50<\/strong><\/p>\n<\/td>\n

\n

75<\/strong><\/p>\n<\/td>\n

\n

100<\/strong><\/p>\n<\/td>\n

\n<\/td>\n<\/tr>\n
\n

Std. Ascorbic acid<\/p>\n<\/td>\n

\n

38.33\u00b10.02<\/p>\n<\/td>\n

\n

45.1\u00b10.04<\/p>\n<\/td>\n

\n

52.95\u00b10.00<\/p>\n<\/td>\n

\n

62.89\u00b10.03<\/p>\n<\/td>\n

\n

66.26\u00b10.05<\/p>\n<\/td>\n

\n

40.26<\/p>\n<\/td>\n<\/tr>\n

\n

Leaves<\/p>\n<\/td>\n

\n

6.71\u00b10.07<\/p>\n<\/td>\n

\n

11.69\u00b10.04<\/p>\n<\/td>\n

\n

17.62\u00b10.01<\/p>\n<\/td>\n

\n

26.93\u00b10.05<\/p>\n<\/td>\n

\n

32.79\u00b10.07<\/p>\n<\/td>\n

\n

49.64<\/p>\n<\/td>\n<\/tr>\n

\n

Stem<\/p>\n<\/td>\n

\n

7.55\u00b10.07<\/p>\n<\/td>\n

\n

10.55\u00b10.05<\/p>\n<\/td>\n

\n

15.2\u00b10.05<\/p>\n<\/td>\n

\n

24.92\u00b10.04<\/p>\n<\/td>\n

\n

30.83\u00b10.02<\/p>\n<\/td>\n

\n

49.89<\/p>\n<\/td>\n<\/tr>\n

\n

Stem bark<\/p>\n<\/td>\n

\n

15.93\u00b10.07<\/p>\n<\/td>\n

\n

29.05\u00b10.01<\/p>\n<\/td>\n

\n

34.92\u00b10.01<\/p>\n<\/td>\n

\n

40.94\u00b10.03<\/p>\n<\/td>\n

\n

51.59\u00b10.03<\/p>\n<\/td>\n

\n

48.85<\/p>\n<\/td>\n<\/tr>\n

\n

Roots<\/p>\n<\/td>\n

\n

18.74\u00b10.04<\/p>\n<\/td>\n

\n

23.07\u00b10.01<\/p>\n<\/td>\n

\n

29.08\u00b10.02<\/p>\n<\/td>\n

\n

34.98\u00b10.01<\/p>\n<\/td>\n

\n

52.64\u00b10.01<\/p>\n<\/td>\n

\n

47.25<\/p>\n<\/td>\n<\/tr>\n

\n

Fruits<\/p>\n<\/td>\n

\n

19.13\u00b10.02<\/p>\n<\/td>\n

\n

25.13\u00b10.03<\/p>\n<\/td>\n

\n

34.82\u00b10.07<\/p>\n<\/td>\n

\n

41.19\u00b10.03<\/p>\n<\/td>\n

\n

52.76\u00b10.06<\/p>\n<\/td>\n

\n

46.36<\/p>\n<\/td>\n<\/tr>\n

\n

Fruit Callus<\/p>\n<\/td>\n

\n

26.29\u00b10.04<\/p>\n<\/td>\n

\n

34.16\u00b10.03<\/p>\n<\/td>\n

\n

36.96\u00b10.05<\/p>\n<\/td>\n

\n

43.18\u00b10.05*<\/p>\n<\/td>\n

\n

58.76\u00b10.01**<\/p>\n<\/td>\n

\n

43.19**<\/p>\n<\/td>\n<\/tr>\n

\n

Suspension Culture<\/p>\n<\/td>\n

\n

27.97\u00b10.02<\/p>\n<\/td>\n

\n

39.53\u00b10.02<\/p>\n<\/td>\n

\n

42.27\u00b10.06*<\/p>\n<\/td>\n

\n

50.19\u00b10.01*<\/p>\n<\/td>\n

\n

60.5\u00b10.05**<\/p>\n<\/td>\n

\n

41.81**<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are given as mean \u00b1 S.D. (n=3).** Significant at p\u02c20.01, * Significant at p\u02c20.05. p-value was calculated by comparing with control by ANOVA followed Dunnett\u2019s test, values are expressed as \u00b1 SEM.<\/p>\n\n\n\n
\"\"<\/td>\n\n

Figure 5: <\/strong>H2<\/sub>O2 <\/sub>radical scavenging<\/strong> of standard and<\/strong> the methanolic extracts o<\/strong>f<\/strong> N. nimmoniana<\/em><\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 6: H2<\/sub>O2 <\/sub>radical scavenging activity and IC 50 (\u00b5g\/ml).<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Antibacterial activity of the\nplant extracts18<\/sup><\/strong><\/p>\n\n\n\n

Well Diffusion\nTechnique<\/strong><\/p>\n\n\n\n

The antibacterial activity was determined against strains of Gram-negative bacteria such as E. coli, Staphylococcus aureus, Klebsiella pneumoniae,<\/em> and Pseudomonas aeruginosa<\/em>. The results revealed that all plant extracts exhibited various levels of potential efficiency in inhibiting the growth of bacteria. The zones of inhibition as observed on the plates revealed that suspension culture and Fruit callus culture showed significant inhibitory activity against E. coli and Staphylococcus aureus<\/em>, moderate inhibitory activity against Pseudomonas aeruginosa, and the<\/em> lowest inhibitory activity against Klebsiella pneumoniae<\/em>. The standard Amoxicillin results in a 30 mm, 37 mm, 35 mm, and 17 mm zone of inhibition against E. coli<\/em>, Staphylococcus aureus, Klebsiella pneumonia, <\/em>and Pseudomonas aeruginosa<\/em>. The suspension culture showed good inhibition zones of 27 mm, 30 mm, and 15 mm, whereas the fruit callus culture showed good inhibition zones of 25 mm, 28 mm, and 15mm against E. coli, Staphylococcus aureus, <\/em>and Pseudomonas aeruginosa<\/em>. Only fruit extracts showed a good inhibition zone of 28 mm against Klebsiella pneumonia,<\/em> as shown in Table 6. (Figure 7-10)<\/p>\n\n\n\n

Table 6: Zone of Inhibition: Using different part of Nothopodytes nimmoniana<\/em><\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

 <\/strong><\/p>\n

Test Samples<\/strong><\/p>\n<\/td>\n

\n

Zone of Inhibition in mm<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

E.coli<\/em><\/strong><\/p>\n<\/td>\n

\n

S. aureus<\/em><\/strong><\/p>\n<\/td>\n

\n

K. pneumonie<\/em><\/strong><\/p>\n<\/td>\n

\n

P. aeruginosa<\/em><\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Amoxicillin (Standard)<\/strong><\/p>\n<\/td>\n

\n

30\u00b10.0<\/p>\n<\/td>\n

\n

37\u00b10.0<\/p>\n<\/td>\n

\n

35\u00b10.0<\/p>\n<\/td>\n

\n

17\u00b10.0<\/p>\n<\/td>\n<\/tr>\n

\n

Leaves<\/strong><\/p>\n<\/td>\n

\n

21\u00b10.0<\/p>\n<\/td>\n

\n

14\u00b10.0<\/p>\n<\/td>\n

\n

23\u00b10.0<\/p>\n<\/td>\n

\n

10\u00b10.0<\/p>\n<\/td>\n<\/tr>\n

\n

Stem<\/strong><\/p>\n<\/td>\n

\n

19\u00b10.0<\/p>\n<\/td>\n

\n

20\u00b10.0<\/p>\n<\/td>\n

\n

22\u00b10.0<\/p>\n<\/td>\n

\n

12\u00b10.0<\/p>\n<\/td>\n<\/tr>\n

\n

Stem bark<\/strong><\/p>\n<\/td>\n

\n

18\u00b10.0<\/p>\n<\/td>\n

\n

17\u00b10.0<\/p>\n<\/td>\n

\n

20\u00b10.0<\/p>\n<\/td>\n

\n

11\u00b10.0<\/p>\n<\/td>\n<\/tr>\n

\n

Roots<\/strong><\/p>\n<\/td>\n

\n

20\u00b10.0<\/p>\n<\/td>\n

\n

14\u00b10.0<\/p>\n<\/td>\n

\n

19\u00b10.0<\/p>\n<\/td>\n

\n

11\u00b10.0<\/p>\n<\/td>\n<\/tr>\n

\n

Fruits<\/strong><\/p>\n<\/td>\n

\n

23\u00b10.0<\/p>\n<\/td>\n

\n

25\u00b10.0<\/p>\n<\/td>\n

\n

28\u00b10.0<\/p>\n<\/td>\n

\n

12\u00b10.0<\/p>\n<\/td>\n<\/tr>\n

\n

Fruit Callus <\/strong><\/p>\n<\/td>\n

\n

25\u00b10.0<\/p>\n<\/td>\n

\n

28\u00b10.0<\/p>\n<\/td>\n

\n

22\u00b10.0<\/p>\n<\/td>\n

\n

14\u00b10.0<\/p>\n<\/td>\n<\/tr>\n

\n

Suspension Culture<\/strong><\/p>\n<\/td>\n

\n

27\u00b10.0<\/p>\n<\/td>\n

\n

30\u00b10.0<\/p>\n<\/td>\n

\n

20\u00b10.0<\/p>\n<\/td>\n

\n

15\u00b10.0<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 7: Zone of Inhibition Leaves extracts and Stem bark extracts against (A)E. coli (B) Staphylococcus aureus (C) Klebsiella pneumoniae (D) Pseudomonas<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 8: Zone of Inhibition Fruits extracts and Stem extracts against (A) E. coli (B) Staphylococcus aureus (C) Klebsiella pneumoniae (D) Pseudomonas.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 9: Zone of Inhibition Roots and Fruit<\/strong> Callus extracts against (A) E. coli.
(B) Staphylococcus aureus (C) Klebsiella pneumoniae (D) Pseudomonas<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 10: Zone of Inhibition suspension culture extracts against (A) E. coli (B) Staphylococcus aureus (C) Klebsiella pneumoniae (D) Pseudomonas<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Determination of\nMinimum inhibitory concentrations (MIC) and <\/strong>Minimum\nbactericidal concentration (MBC)<\/strong><\/strong><\/p>\n\n\n\n

The standard and test sample concentrations for MIC were 0.1 mg\/ml, 0.5 mg\/ml, 1mg\/ml, 2mg\/ml, and 5mg\/ml. The suspension culture sample shows the minimum inhibition concentration for E. coli (<\/em>2 mg\/ml), Staphylococcus aureus (<\/em>0.1 mg\/ml), Klebsiella (<\/em>2 mg\/ml), and Pseudomonas (<\/em>0.1 mg\/ml). The suspension culture sample also showed MBC for E. coli (5 mg\/ml) and Staphylococcus aureus (<\/em>5 mg\/ml). The Fruit callus sample shows the minimum inhibition concentration for E. coli (<\/em>2 mg\/ml), Staphylococcus aureus (<\/em>2 mg\/ml), Klebsiella (2<\/em> mg\/ml), and Pseudomonas (<\/em>2 mg\/ml). The Fruit callus sample also showed MBC for E. coli (5 mg\/ml) and Staphylococcus aureus (<\/em>5 mg\/ml). Among the four bacterial strains, E. coli<\/em> and Staphylococcus aureus <\/em>showed great potency at low concentrations of the drug compound. Comparatively to suspension culture and fruit callus culture, the other plant extracts show the minimum inhibition concentration (MIC) for all four pathogens at high concentrations, as summarised in Table 7.<\/p>\n\n\n\n

Table 7: Determination of Minimum inhibitory Concentration (MIC) and Minimum bactericidal concentration (MBC)<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Test Samples<\/strong><\/p>\n<\/td>\n

\n

MIC (<\/strong>m<\/strong>g\/mL)<\/strong> <\/strong><\/p>\n<\/td>\n

\n

MBC <\/strong>(<\/strong>m<\/strong>g\/mL) <\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

E. coli<\/em><\/strong><\/p>\n<\/td>\n

\n

S. aureus<\/em><\/strong><\/p>\n<\/td>\n

\n

K. pneumonie<\/em><\/strong><\/p>\n<\/td>\n

\n

P. aeruginosa<\/em><\/strong><\/p>\n<\/td>\n

\n

E. coli<\/em><\/strong><\/p>\n<\/td>\n

\n

S. aureus<\/em><\/strong><\/p>\n<\/td>\n

\n

K. pneumonie<\/em><\/strong><\/p>\n<\/td>\n

\n

P. aeruginosa<\/em><\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Amoxicillin (Std)<\/strong><\/p>\n<\/td>\n

\n

2mg\/ml<\/p>\n<\/td>\n

\n

2mg\/ml<\/p>\n<\/td>\n

\n

1mg\/ml<\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Leaves<\/strong><\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

1mg\/ml<\/p>\n<\/td>\n

\n

2mg\/ml<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Stem<\/strong><\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

2mg\/ml<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Stem bark<\/strong><\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

2mg\/ml<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Roots<\/strong><\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Fruits<\/strong><\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Fruit Callus<\/strong><\/p>\n<\/td>\n

\n

2mg\/ml<\/p>\n<\/td>\n

\n

1mg\/ml<\/p>\n<\/td>\n

\n

2mg\/ml<\/p>\n<\/td>\n

\n

2mg\/ml<\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

2mg\/ml<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Suspension Culture<\/strong><\/p>\n<\/td>\n

\n

2mg\/ml<\/p>\n<\/td>\n

\n

0.1mg\/ml<\/p>\n<\/td>\n

\n

2mg\/ml<\/p>\n<\/td>\n

\n

0.1mg\/ml<\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

5mg\/ml<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Antifungal activity<\/strong><\/p>\n\n\n\n

The standard Fluconazole resulted in a 27-mm zone of\ninhibition against Candida albicans. <\/em>The\nsuspension culture and Fruit callus exhibited good inhibition zones of 24 mm\nand 21 mm, whereas leaves, stem, stem bark, roots, and fruit extract showed\ninhibition zones of 10 mm, 18 mm, 14 mm, 19 mm, and 15mm. Summarised in table\nno. 8.<\/p>\n\n\n\n

Table 8: Zone of Inhibition: Using different part of Nothopodytes nimmoniana.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Test Samples<\/strong><\/p>\n<\/td>\n

\n

Zone of Inhibition in mm<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Candida Albicans<\/em><\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Fluconazole<\/strong> (Standard)<\/strong><\/p>\n<\/td>\n

\n

27\u00b10.0<\/p>\n<\/td>\n<\/tr>\n

\n

Leaves<\/strong><\/p>\n<\/td>\n

\n

10\u00b10.0<\/p>\n<\/td>\n<\/tr>\n

\n

Stem<\/strong><\/p>\n<\/td>\n

\n

18\u00b10.0<\/p>\n<\/td>\n<\/tr>\n

\n

Stem bark<\/strong><\/p>\n<\/td>\n

\n

14\u00b10.0<\/p>\n<\/td>\n<\/tr>\n

\n

Roots<\/strong><\/p>\n<\/td>\n

\n

19\u00b10.0<\/p>\n<\/td>\n<\/tr>\n

\n

Fruits<\/strong><\/p>\n<\/td>\n

\n

15\u00b10.0<\/p>\n<\/td>\n<\/tr>\n

\n

Fruit Callus<\/strong><\/p>\n<\/td>\n

\n

21\u00b10.0<\/p>\n<\/td>\n<\/tr>\n

\n

Suspension Culture<\/strong><\/p>\n<\/td>\n

\n

24\u00b10.0<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The\nzones of inhibition as observed in the plates revealed that N. nimmoniana <\/em>Fruit callus and suspension culture were shown good inhibitory\nactivity against Candida\nalbicans. <\/em>Whereas\nstem and roots extracts showed activity against Candida albicans<\/em> (Shown in Figure 11).<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 11: Zone of Inhibition extracts of leaves (A), stem (B), roots (C), stem bark (D), fruits (E), fruit callus (F), and suspension culture (G), extracts against Candida Albicans<\/em><\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Determination\nof Minimum inhibitory concentrations (MIC) and <\/strong>Minimum\nfungicidal concentration (MFC)<\/strong> <\/strong><\/p>\n\n\n\n

The standard and\ntest sample concentrations for MIC were 0.5 mg\/ml, 1 mg\/ml, 2mg\/ml, 4 mg\/ml,\nand 6mg\/ml. The minimum inhibitory concentration (MIC) for Candida albicans<\/em> is shown in the leaf extract\n(6 mg\/ml), stem extract (6 mg\/ml), stem bark extract (6 mg\/ml), root extract\n(4mg\/ml, fruit extract (4mg\/ml, callus sample (4 mg\/ml), and suspension culture\nsample (2 mg\/ml). The minimum fungicidal concentration (MFC) for Candida albicans<\/em> shown in fruit extracts and\nfruit callus samples is 6 mg\/ml. Table No. 9<\/p>\n\n\n\n

Table 9: Determination of Minimum inhibitory Concentration (MIC) and Minimum fungicidal concentration (MFC)<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Test Samples<\/strong><\/p>\n<\/td>\n

\n

MIC (<\/strong>m<\/strong>g\/ml) <\/strong><\/p>\n<\/td>\n

\n

MFC <\/strong>(<\/strong>m<\/strong>g\/ml) <\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Candida Albicans<\/em><\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Fluconazole<\/strong> (Standard)<\/strong><\/p>\n<\/td>\n

\n

4mg\/ml<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Leaves<\/strong><\/p>\n<\/td>\n

\n

6mg\/ml<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Stem<\/strong><\/p>\n<\/td>\n

\n

6mg\/ml<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Stem bark<\/strong><\/p>\n<\/td>\n

\n

6mg\/ml<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Roots<\/strong><\/p>\n<\/td>\n

\n

4mg\/ml<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Fruits<\/strong><\/p>\n<\/td>\n

\n

4mg\/ml<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Fruit Callus<\/strong><\/p>\n<\/td>\n

\n

4mg\/ml<\/p>\n<\/td>\n

\n

6 mg\/ml.<\/p>\n<\/td>\n<\/tr>\n

\n

Suspension Culture<\/strong><\/p>\n<\/td>\n

\n

2mg\/ml<\/p>\n<\/td>\n

\n

6 mg\/ml.<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Conclusion <\/strong><\/p>\n\n\n\n

In this research,\nit was observed that Nothopodytes nimmoniana<\/em>\nmethanolic extracts were obtained from the different plant parts and from in\nvitro callus and plant cell suspensions. All extracts showed antioxidant and\nantimicrobial activity, but fruit callus and suspension culture <\/em>showed the highest antioxidant and\nantimicrobial properties. The reducing power of the suspension culture and\nfruit callus culture was enhanced with the increase in concentrations. The\nfruit callus culture and cell suspension culture showed higher concentrations\nof total phenolics and flavonoids, contributing to antioxidant and\nantimicrobial activity compared to normal extracts of other parts of the plant.\nThis study indicates that the plant fruit and suspension cultures contain\nincreased concentrations of compounds contributing to antioxidant and\nantimicrobial activity, which could be used for MDR bacterial and fungal\ninfections and the development of useful natural compounds.<\/p>\n\n\n\n

Acknowledgments<\/strong><\/p>\n\n\n\n

Authors would like to express our sincere gratitude and thanks to Amity University, Madhya Pradesh, Gwalior, for their constant support, guidance, and successfully completing this research work,<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

there is no conflict of interest.<\/p>\n\n\n\n

Funding Source:<\/strong><\/p>\n\n\n\n

Acknowledge MPCST, Bhopal, India, project number R&D\/RP-2\/2018-10\/273, for giving financial support.<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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