Quantitative Phytochemical Analysis Reveals significant Antibiofilm Activity in Pleione maculata, an endangered Medicinal Orchid

Pleione maculata has no scientific reports on quantitative phytochemical and antibiofilm activity till date. the objective of the study was to quantify and determine medicinally important bioactivity in P. maculata and analyse its anti-biofilm activity against clinical isolates Staphyloccocus aureus, Klebsiella pneumoniae and Proteus mirabilis. P. maculata exhibited the highest total Antioxidant Capacity (tAC) about 193.98±0.1 mg, highest total Phenolic Content (tPC) at 552± 0.0 mg and total Flavonoid Content (tFC) were observed highest at 879.5±0.2 mg. the acetone and ethyl acetate extracts of P. maculata pseudobulb showed distinct and significant zone of inhibition (ZOi) against drug-resistant S.aureus about 16±0.00 mm (MiC 0.875 mg/ml), ZOi of acetonitrile pseudobulb extract against P. mirabilis was 15.33±0.4 mm (MiC 1 mg/ml), ZOi of acetonitrile extracts of leaves and stem, ethyl acetate extract of pseudobulb was 12±0.0 mm, 12±01.4 mm, 12±2.8 mm against K. pneumoniae (MiC 1.8 mg/ml, 0.68 mg/ ml and 3 mg/ml). Acetonitrile extract of pseudobulbs exhibited the highest Minimum Biofilm inhibition concentration (MBiC) at 0.25 mg/ml against S. aureus, water root extract inhibited attachment of K. pneumoniae with lowest MBiC value 0.093 mg/ml, water and acetone extract of leaves inhibited cell attachment of P. mirabilis at lowest MBiC 0.117 and 0.171 mg/ml. the UV-Vis absorption band of P. maculata extracts ranges from 204-665 nm indicating the presence of phenolic and flavonoid compounds. the study indicates the potentiality of P. maculata as a rich source of medicinal active compounds as an antibiofilm agent against antibiotic-resistant clinical isolates.

various biological effects, including free radical scavenging, antioxidant activity, and anti-tumor activity 15 .
Today, the science of dealing with the prevention and treatment of diseases caused by microorganisms has come down due to some antibiotic-resistant bacteria. The resistance may be due to the wide use of antibiotics as growth promoters in animal food and maybe overuse of antibiotics in humans 1 . Traditional treatment processes against bacterial infections depend on different antimicrobial compounds or antibiotics that can either inhibit or kill the growing microbial cells 17 . But pathogenic microorganisms can resist themselves against those inhibitory compounds by forming microbial communities termed biofilms 16 . Biofilms are the assemblages of microbial cells embedded in a matrix of self-produced polymeric substances consisting of polysaccharides, proteins, and DNA. The transcribed genes of biofilm-associated microorganisms differ from their planktonic cells 18 . Adhesion, followed by microcolony development, maturation, and dispersion, are the four distinct phases of microbial biofilm formation 19 . The dispersal mechanism is critical because it starts a new infection cycle by the colonization of a new surface, allowing microbial infection to spread quickly 20 . Biofilms adhere to biological and nonbiological medical devices, urinary catheters, living tissue, dental and industrial setting surfaces 21 . The biofilm has a slower growth rate and secretes various surface chemicals and virulence factors, increasing their toxicity by hundreds of times 22 . Microbial biofilms are involved in several chronic infectious diseases in humans, an estimated 65% of all infections according to the Centre for disease control (CDC) and 80% according to the National Institutes of Health (NIH) 16 . Urinary tract infections, pneumonia, cystic fibrosis, periodontitis, endocarditis, osteomyelitis, rhinosinusitis are caused by bacterial biofilm 24 . Several bacteria are highly pathogenic to humans. Staphylococcus aureus is a pathogen causing various diseases on the human host through biofilm formation 25 and is multidrug-resistant 26,27 . Klebsiella pneumoniae isolated from urine, sputum, or wound swabs can form up to 40% biofilm. That can cause nosocomial infections such as urinary tract infections, septicemia, pneumonia, and tissue infections 26 .
The Gram-negative rod-shaped bacteria Proteus mirabilis is the most common cause of community and healthcare-associated illnesses. Multidrugresistant (MDR) strains of P. mirabilis can cause severe complications in the survival of hospitalized patients 28 . Thus, effective strategies can combat these biofilm-linked diseases caused by different microbes. A phytochemical analysis can examine quantitative phytoconstituents in plants to assess plant characteristics. There are few techniques and give a brief idea for identifying the compounds. The most popular ones are spectroscopic and chromatographic techniques. A simple, costeffective, and sensitive technique is UV-Visible spectroscopy 29 . There are no scientific reports on total antioxidant activity, total phenolics, and total flavonoid content of Pleione maculata, and the antibiofilm activity against clinical isolates.
The study aims to evaluate the bioactive phytochemicals in Pleione maculata. Also, to assess the antibiofilm activity against select antibiotic-resistant isolates. The target isolates are Staphylococcus aureus (Gram-positive), Klebsiella pneumoniae (Gram-negative), and Proteus mirabilis (Gram-negative).

MAteRiAls AND MethODs Collection, preparation, and extraction of phytocompounds
Pleione maculata (Fig. 1.) was collected in Khliehriat, East Jaintia Hills District, Meghalaya, India, during the spring season (temperature was 19°C). For the plant sample, herbarium sheets were prepared. The leaves, pseudobulbs, stems, and roots of P. maculata were weighed individually after being surface sterilized with 70% ethanol. Plant parts were separated and dried in the shade for two weeks. Using a household grinder, the dry materials were ground into a fine powder and kept in microcentrifuge tubes at 4˚C 30 . The powdered plant material was weighed and soaked in a 1:45 (w/v) ratio in several solvent systems (acetone, acetonitrile, chloroform, ethanol, ethyl acetate, methanol, water). The extracts were agitated for 48 hours at 37°C in an incubator shaker 31 . Extracts were filtered using Whatman filter paper No. 1 after incubation. For the remaining fractions, filtration was repeated. Evaporation at a constant temperature of 30°C concentrated the extracts. The concentration of extracts obtained is listed in Table 1. Antioxidant activity Determination of antioxidant activity by 2, 2-diphenyl-1-picrylhydrazyl (DPPh) radical scavenging activity Each extract (leaves, pseudobulb, stems, and roots) of Pleione maculata was tested to determine the radical scavenging activity 36 . For this, 2,2-diphenyl-1-picryl-hydrazyl (DPPH) (HiMedia) Journal of Pure and Applied Microbiology solution was prepared at a concentration of 1 mg/ mL with methanol. The mixture was set in the dark. A stock concentration (1mg/mL) of plant extracts was prepared. The plant extracts and the standard L-ascorbic acid (HiMedia) were diluted at different concentrations (20,40,60,80, 100 µg/mL). A 0.1 mL methanolic DPPH solution was combined with varying concentrations of test extracts on a 96-well microtiter plate. The solution was mixed thoroughly and incubated in the dark at room temperature for 30 minutes. The colour reduction of DPPH was measured at 520 nm in BioEra Life Science Microplate reader Model Nova. A standard curve was plotted with absorbance against the concentration of extracts. The blank and control OD was measured. DPPH free radical scavenging activity can be determined using the formula: The test was performed in triplicates and a graph was plotted with the percentage of DPPH radical scavenging activity against the concentration of samples extracts in µg/mL. IC 50 values were calculated using statistical functions in Microsoft excel 2010.

Determination of total antioxidant capacity by Molybdate Assay
The total antioxidant activity was measured using a modified molybdate assay 37 . The test relies on the reduction of Mo (VI) to Mo (V) after the creation of a complex greenblue phosphate/Mo (V) complex at an acid pH. Molybdate reagent solution was prepared using the ascorbic acid standard solution at various concentrations (20, 60, 80, and 100 mg/mL). The plant extracts were made with dimethyl sulphoxide (DMSO) (HiMedia) at a concentration of 2 mg/mL. A 400µl of plant extract was pipetted out and diluted with methanol, 1.5 mL of molybdate reagent solution was added, and test tubes were sealed with aluminum foil before incubation in water bath at 95°C for 90 minutes. At room temperature, the mixture was allowed to cool. An SYSTRONICS PC-based double beam UV-VIS spectrophotometer-2202 was used to measure the absorbance at 695nm. The blank was made by combining 400 µl of the solvent used for sample extraction with 1.5 mL of molybdate reagent.
The total antioxidant capacity was measured in grams of ascorbic acid equivalents. The antioxidant activity was measured in milligrams of ascorbic acid equivalents (AAE) per milliliter of solution.

Determination of total phenolic content by Folin Ciocalteau assay
Folin Ciocalteau test 38 with modification was used to measure the total phenolic content in each extract. Plant extracts were diluted in 3 mL of DMSO to achieve a concentration of 2 mg/mL. For the calibration curve, gallic acid (3, 4, 5-Trihydroxy benzoic acid -SRL) was collected at various concentrations (20,40,60,80, 100, 120, 140, and 160 mg/mL) 39 . A 400µl of the test extracts (leaves, pseudobulb, stems, and roots) were mixed with 100µl ethanol and 0.2 mL of Folin Ciocalteau reagent (MERCK-DJ4D640513). The final volume was adjusted with distilled water. Then, 1 mL of 7% sodium carbonate solution was added. The tubes were vortexed and incubated in the dark at room temperature for 2 hours. Using an SYSTRONICS PC-based double beam UV-VIS spectrophotometer-2202, the mixture's absorbance was measured at 760 nm. The total phenolic content (TPC) was expressed in milligrams of Gallic acid equivalent per gram of P. maculata extracts (mg GAE/g of P. maculata extracts). The following formula was used to determine the total phenolic content of the extract with gallic acid equivalent 12 . Blank was prepared by adding only methanol in equal volume Determination of total flavonoid content by Aluminium chloride method The AlCl 3 technique with modification was used to determine the total flavonoid content of various plant extracts 38 . Plant extracts were prepared at a concentration of 2 mg/mL. A standard quercetin solution was prepared by pipetting 5 mL of stock and diluting it to make a final volume of 10 mL with 80 percent ethanol. For the calibration curve, a reference solution of quercetin (HiMedia-RM6191-25G) was obtained at various concentrations (20,40,60, 80, 100, 120, 140, and 160 mg/mL). A 400µl of the test extracts (leaves, pseudobulb, stems, and roots) and standard quercetin solution were diluted with ethanol. To this, 0.3 mL of 5% NaNO3, 0.3 mL of 10% aluminium chloride solution, and 1M NaOH solution were added. The final volume was adjusted with distilled water. The tubes were vortexed and incubated at room temperature for 40 minutes. Using an SYSTRONICS PC-based double beam UV-VIS spectrophotometer-2202, the mixture's absorbance was measured at 510 nm. The total flavonoid content (TFC) of Pleione maculata extracts was measured in mg quercetin equivalent per gram of extracts (mg Quercetin/g of P. maculata extracts). The following formula was used to determine the total flavonoid content in the extract with quercetin equivalent 12 .

Preparation of inoculums
The overnight microbial culture was prepared using Luria Bertani broth (Miller-HiMedia M1245-1KG) at 37±2˚C. A 40µl of the microbial culture was diluted in Eppendorf containing 1 mL of sterile double distilled water 33 . A 0.5 McFarland turbidity standard is a reference used to adjust the inoculum density at 1.5 X 108 CFU/mL 34 .

Antibacterial activity by Kirby-Bauer Disc diffusion susceptibility method
Antibacterial activity was tested by the disc diffusion method using a 6mm disc. A Luria Bertani agar (Miller-HiMedia M1151-500G) was prepared and sterilized in an autoclave for 15 minutes at 121˚C. The standard working inoculums were swab using a sterile L-shaped glass spreader at an angle of 60˚C with three times rotation 34 . The discs were dipped in different extracts and dried, followed by inoculation in respected microbial plates. The plates were incubated at 37±2 ˚C for 24 hours in an incubator. The zone of inhibition was observed the next day and measured in millimetres (including 6 mm diameter of the disc). The experiments were performed in duplicates. The ZOI was compared with standard reference antibiotic 35 .

Broth micro-dilution method (MiC) with Resazurin indicator
MIC is the lowest concentration of extract required to inhibit the growth of microbes in comparison with non-treated (negative) control. Overnight clinical isolates suspension was incubated at 37˚C for 24 hours. Initial suspension was diluted (1:20 ratio) to adjust optical density with 0.5 McFarland's standards (0.5 OD). Luria Bertani broth was added to a sterile 96-wells microtitre plate containing test extracts followed by serial dilution. To 10µL of the inoculum in each well, 10µL of resazurin indicator (7-hydroxy-3H-phenoxazin-3-one-10-oxide sodium salt) (HiMedia RM125-1G) was added. A negative control containing 10% DMSO with inoculated medium, and a positive control containing antibiotics with the inoculated medium were also prepared. The 96-well plates were sealed with parafilm and incubated at 37±2˚C for 24 hours. The experiment was repeated in duplicates. The lowest concentration was the MIC for the extracts with inhibited growth of test organisms 48 .

Antibiofilm assay screening biofilm formation ability of the test organisms (tube method)
Clinical isolates (S. aureus, K. pneumoniae, and P. mirabilis) were tested via biofilm screening conducted using the tube method with minor modifications 47 . To do so, 10 µl loopful of overnight bacterial cultures were added to tubes containing 5 mL of Luria-Bertani (LB) Broth and incubated at 37±2˚C for 24-48 hours. After the incubation period, the tubes were washed off three times using sterile double-distilled water and dried prior to staining. The tubes were then stained with 0.1% (v/v) crystal violet and allowed to stand for 10 minutes. Subsequently, the tubes were washed off and the excess stain was removed, followed by drying at room temperature to observe biofilm formation. The stained was re-dissolved in 30% (v/v) glacial acetic acid and absorbance was measured at 492 nm using a SYSTRONICS PC-based double beam UV-VIS spectrophotometer-2202 with glacial acetic acid suspension.

Minimum Biofilm inhibitory Concentration (MBiC) of initial cell attachment assay
The minimum inhibitory concentration of test plant extracts was estimated by ten-fold serial dilution in 96-well polystyrene plates. From fresh LB broth, 100 µl was pipetted to each well of a microtiter plate, and 50 µl of overnight cultured biofilm-forming isolates added. Positive control contained bacteria with the reference antibiotic, and negative control was bacteria without antibiotics prepared for each isolate for comparison. The plates were then sealed with a parafilm and kept for 24-48 hours incubation at 37±2˚C to check the potentiality of test plant extracts to inhibit initial cell attachment of biofilm-forming clinical isolates. The plates were washed and stained with 0.1% (v/v) crystal violet for 10 minutes, and the excess stain was removed with double distilled water and dried at room temperature. Absorbance was measured at 492nm using a BioEra Life science Microplate reader Model Nova. The percentage of biofilm inhibition was compared with untreated control.
Percentage biofilm inhibition was calculated using the following formula: OD of untreated control

Preparation of extracts for UV-Vis spectroscopy analysis
Dried plant samples weighing 0.25g were soaked in 5mL different solvents (acetone, acetonitrile, chloroform, ethanol, ethyl acetate, methanol, and water) 40 , and were incubated in a shaker for 24 hours. The extracts were centrifuged at 3000 rpm for 10 minutes and filtered using Whatman filter paper No. 1 qualitative circle 125mm diameter. The filtrates were scanned ranging from 200-1100nm in a spectrophotometer 29 .

statistical analysis
Experiments were conducted in duplicates, triplicates, and experimental data were presented as mean values ± SD. The concentration of IC50 that could inhibit 50% DPPH free radical was calculated using statistical Microsoft excel 2010. Using IBM SPSS Ver 20 Multivariate statistical analysis viz. Hierarchical cluster analysis (HCA) was performed. The relationship among different samples was presented as clades of a dendrogram which measured Squared Euclidean distance (clustered by the average linkage between groups) 41 .

ResUlts
The finely powdered sample of P. maculata was solvent extracted. The antioxidant activity, quantitative phytochemical analysis, and antibiofilm activity were determined using the solvent extracts. Preliminary compounds were identified using SYSTRONICS PC-based double beam UV-VIS spectrophotometer-2202. the fresh and dry weight of different parts of P. maculata The length of P. maculata leaves measured 15.5 cm in matured leaves with a surface area of 2.3 cm width; pseudobulb length measured 2.3 cm with green and purplish colour. It gave an aroma of locally prepared medicine. Table 2 lists the differences between the fresh and dry weights. For instance, fresh pseudobulb weighed about 200.59 grams, whereas 27.6 grams in dry weight. It indicates high water storage and mineral content in pseudobulb.

Antioxidant activity
Antioxidant activity using DPPH radical The extracts of P. maculata were able to decolorize the deep purple color of DPPH radical to yellow color. This indicates the capability of extracts to scavenge and neutralize free radicals. Acetone and acetonitrile are suitable solvents for the extraction of antioxidant compounds present in P. maculata. The capability to neutralize DPPH harmful free radicals indicates extracts' potentiality as an antioxidant source attributed to the presence of a high amount of polyphenols 42 . The standard ascorbic acid showed no significant difference between the extracts of P. maculata. The different solvent extracts of P. maculata can be a promising source of antioxidant compounds with the highest scavenging activity of about 89.583% observed in acetone extract of pseudobulb, 89.216 % in methanol extract of pseudobulb, 88.235% in acetone extract of leaves, 82.598% in reference ascorbic acid standard as presented in Fig. 2 The IC 50 value calculated was found lowest in acetone extract of leaves (20.76±0.00 µg/mL), acetonitrile extract of the stem (22.30±0.00 µg/ mL), acetone extract of pseudobulb (23.03±0.00 µg/mL) which is lower than ascorbic acid standard (30.80±0.01µg/mL). The lower the IC 50 concentration value than the ascorbic acid standard, the higher the antioxidant activity of samples. The observed IC 50 value of different extracts of P. maculata was lower in acetone extracts than other solvent extracts represented in Table 3.
TAC was calculated using the linear equation obtained by plotting the absorbance value of standard ascorbic acid against the concentration. The equation was y = 0.0241x + 0.1176 with R2 = 0.9677 where 'y' is absorbance of extracts, and 'x' is Total Ascorbic acid equivalent (TAE) in mg per grams of extracts. Total antioxidant capacity was highest in ethanol, ethyl acetate, methanol, and acetone extracts of P. maculata ( Table 4). The TAC of ethyl acetate extract of leaves was 193.98±0.1 mg of TAE/gram of extracts, 198.14±0.3 mg of TAE/grams of ethanol extract of leaves. The least antioxidant capacity was 2 mg of TAE/grams of water extracts of roots.

Quantitative phytochemical analysis
The concentration of phenolics in P. maculata extracts was expressed as gallic acid equivalent per gram of the extract using the gallic acid standard calibration curve. The equation obtained by plotting a graph with the absorbance  value of gallic acid standard on the y-axis against concentration on the x-axis was y = 0.0096x + 0.0966 with the R2 = 0.9898. Phytochemical screening was performed for the seven solvents extracts of P. maculata. The solvents were acetone, acetonitrile, chloroform, ethanol, ethyl acetate, methanol, and water. LEA showed the highest phenolic content with 552±0.0 mg of GAE/ grams of extracts listed in Table 5. PAN extracts yielded 509.375±0.0 mg of GAE/ grams of extracts. Total phenolic content was lowest in chloroform (SC) extracts of the stem with 25.48±0.0 mg of GAE/ grams of extracts. Chloroform may not be an ideal solvent choice for phenolic extraction of P. maculata. The concentration of flavonoids in various extracts of P. maculata was expressed as Quercitin equivalent per gram of the extracts using the quercetin calibration curve equation obtained by plotting a graph with absorbance value of the standard quercitin on the y-axis against concentration on the x-axis. The equation obtained was y = 0.0046x + 0.0819 with the R2 = 0.9733.
The total flavonoid content was highest in LA extracts with 879.5±0.2 mg of QE/ grams, and PAN showed 845.2±0.2 mg of QE/ grams as listed in Table 6. Total flavonoid content was the least in SW extracts with 20.40±0.0 mg of QE/ grams of extracts. Leaves and pseudobulb of P. maculata showed better results for antioxidant capacity, phenolics, and flavonoid content.

effect of Pleione maculata against clinical isolates
P. maculata extracts were effective against all three clinical isolates. Table 7 presents the effective zone of inhibition, and Table 8 presents the MIC value of all the tested extracts. The acetone and ethyl acetate extracts of pseudobulb showed the highest diameter of inhibition (16±0.00mm) against S. aureus with a MIC value of 0.875 mg/mL for PA. Acetonitrile extracts of leaves and stem, and ethyl acetate extract of pseudobulb showed inhibition of 12±1.00 mm against K. pneumoniae with MIC value of 1.375 mg/mL for LAN, 0.6875 mg/mL for SAN and, 3 mg/mL for PEA. The acetonitrile extracts of pseudobulb showed a zone of inhibition (ZOI) of about 15.33±0.33 mm with a MIC value of 1 mg/mL against P. mirabilis. Amoxicillin, a reference antibiotic, showed a ZOI of 11.33±0.00 mm against S. aureus and a ZOI of 10.66±0.00 mm against P. mirabilis with a MIC value of 1mg/mL. Amoxicillin did not inhibit K. pneumoniae. Inhibition by amoxicillin was less effective as compared to acetone pseudobulb extract of P. maculata.

screening of biofilm formation on the selected clinical isolates
The clinical isolates screened for biofilm formation were observed to be positive for the three isolates. Gram-positive bacteria S. aureus showed a slightly stained film on the end walls of the test tubes, gram-negative bacteria K. pneumoniae formed a very thick stained film on the sides of the test tubes, and gram-negative bacteria P. mirabilis formed a moderately stained film on the end walls of the tubes.

effect of P. maculata on the inhibition of initial biofilm attachment
The Minimum Biofilm Inhibition Concentration (MBIC) for all extracts is presented in Table 9. Among Pleione maculata extracts, the pseudobulb acetonitrile extract of P. maculata exhibited the highest inhibitory activity against initial cell attachment of clinical isolates at its highest dilution, 10 −5 , with 87% inhibition of S. aureus cell attachment and the lowest MBIC at 0.25mg/mL. At a 10 −6 dilution water extract of root and leaves, pseudobulb acetonitrile extracts inhibited 97%, 90%, and 87% attachment of K. pneumoniae with MBIC values of 0.093, 0.117, and 0.125 mg/mL, respectively. Water extract of leaves, acetone extract of leaves, water root extracts, ethyl acetate leaf extracts, and pseudobulb acetonitrile extracts inhibited 100%, 91%, 89%, 87%, and 86% attachment of P. mirabilis at the lowest dilution, 10 −6 , with MBIC values of 0.117, 0.171, 0.090, 0.097, and 0.125 mg/ mL, respectively. These results indicate that P. maculata is as effective as many known medicinal plants and antimicrobial agents.

UV-Vis spectrophotometer
The results of UV-VIS spectrogram of seven solvents (acetone, acetonitrile, chloroform, ethanol, ethyl acetate, methanol, and water) extracts of P. maculata exhibited absorption band ranging from 204-665 nm, as shown in Fig. 3. The UV-VIS spectrogram results of P. maculata extracts indicate phenolic compounds such as flavonoids, terpenoids, and tannins in the plant extracts. The absorption band ranging from 204-665 nm is a characteristic feature of flavonoids and their derivatives 44,35 . The absorption of flavonoids ranges from 230-285 (Band I) and 300-350 (Band II) 45,35 . There are two absorption bands of flavonoid and terpenoids that range from 230-290 nm (Band I) and range from 400-550 nm (Band II), whereas the range between 600-700 nm represents the absorption of chlorophyll 44 . hierarchical cluster analysis (hCA) hCA between DPPh and total antioxidant capacity Hierarchical Cluster Analysis (HCA) is a multivariate analysis that identifies natural clusters without prior knowledge about the data to provide useful information such as the graphical representation of the resulting partitions in the form of a hierarchy or dendrogram revealing more information 50 . HCA was performed to determine the similarity or dissimilarity of clusters between various extracts of P. maculata to determine the robustness of the analysis. The horizontal axis of a dendrogram indicates the distance or dissimilarity between clusters whereas the vertical axis represents object clusters. In the HCA dendrogram, six individual clusters were grouped as shown in Fig. 4. to form two grand clusters based on two different methods used for the experiments. In Fig. 4. (i)(a), the rows of leaves of DPPH and pseudobulb DPPH are remarkably similar and have low dissimilarity as they are closely clustered at a rescaled distance of 1 unit from each other as compared to the stem DPPH, which also shows some minute similarities at a rescaled distance of 14 units. The root DPPH shows a completely different clade from the leaves DPPH, pseudobulb DPPH, and stem DPPH. In Fig. 4 (i)(b), the rows of leaves TAC and pseudobulb TAC have low dissimilarity as they are closely clustered at a rescaled distance of 2 units from each other and from stem TAC, which also shows some minute dissimilarity at a rescaled distance of 1 unit. The root TAC shows a completely different clade from the leaves TAC, pseudobulb TAC, and stem TAC. hCA between tFC and tPC Fig. 4 illustrates two grand clusters based on two different methods used for the experiments. Six individual clusters were grouped together. In Fig. 4 (ii) (a), the rows of stem TFC and root TFC are very similar as they are closely clustered at a rescaled distance of 1 unit as compared to the pseudobulb TFC, which also shows similarities at a rescaled distance of 3 units. The leaves TFC shows a completely different clade with few similarities in the pseudobulb, stem and root TFC at a rescaled distance of 3 units. In Fig.  4 (ii) (b), the rows of leaves TPC and pseudobulb TPC have low dissimilarity as they are clustered at a rescaled distance of 2 units from each other and from stem TPC, which also shows some minute dissimilarity at a rescaled distance of 3 units. The root TPC shows a completely different clade from leaves TPC, pseudobulb TPC, and stem TPC at a rescaled distance of 6 units. hCA of antibiofilm activity Fig. 5 illustrates the extracts cluster based on similarities in the presence of antibiofilm activity in a series of dilution factors. In Fig. 5 (a), there are five grand clusters of extracts: RW, AMX, and SW form a single cluster with a similar dilution factor 10 −1 at a rescaled distance of 1 unit, PAN and SAN form one cluster with a similar dilution factor 10 −5 at a rescaled distance of 1 unit, and LAN forms one grand cluster with a dilution factor 10 −4 , which indicates large differences with other extracts at a rescaled distance of 23 units. In Fig. 5 (b), the HCAs of various extracts against K. pneumoniae form two grand clusters. Each grand cluster is further divided into two grand clades based on the similarities in their dilution factors. Extracts LM, PM, LA, PA, and SETH fall under the same clade, which indicates similarities in the presence of antibiofilm activity with the same dilution factor 10 −6 at a rescaled distance of 1 unit. LETH extracts fall under one sub-clade alone because of their dissimilarity with other extracts at a rescaled distance of 17 units. In Fig. 5 (c), the HCAs of extracts against P. mirabilis reveal two grand clusters representing two grand clades, with four sub-clades. Extracts PW, SW, and RW are remarkably similar as they belong to the same leaf in a sub-clade with a dilution factor of 10 −6 whereas extract PETH exhibits a single separate grand cluster, which indicates high dissimilarity with other extracts at a rescaled distance of 25 units.

DisCUssiON
Preliminary qualitative phytochemicals were earlier reported in P. maculata collected from Meghalaya, which showed the presence of secondary metabolites such as terpenoid, phenol, tannin, saponin, coumarin, and cardiac glycosides in the leaves, pseudobulb, and seeds 51 . In vitro pseudobulb explants regeneration was conducted for the conservation of the rare and therapeutically important P. maculata 5 . P. maculata collected from Arunachal Pradesh also reported the presence of phytochemicals such as flavonoids, steroids, cardiac glycosides, and alkaloids 52 . The bioactivity of P. maculata was compared mainly with the closely related genus Coelogyne and its various species. A species of Coelogyne stricta exhibited 93% antibiofilm activity 53   to combat them effectively. Pleione maculata, an epiphytic rare medicinal orchid containing numerous effective secondary metabolites, was evaluated to gauge its antimicrobial and antibiofilm activity 51 . The ethanolic pseudobulb extract of Coelogyne speciosa exhibited strong antibacterial activity against S. aureus (ZOI was 19 mm) and methanolic pseudobulb extracts (ZOI was 8 mm), whereas ethyl acetate extracts showed no bacterial inhibition 56 . The extracts of Coelogyne stricta showed no antibacterial activity against S. aureus. The ethanolic extracts of Coelogyne brachyptera showed good antibacterial activity with a ZOI of 20 mm against gram-positive S. aureus 56 . It has also exhibited antibacterial activity against S. aureus with a strong ZOI of 17±0.6 mm 52 . Coelogyne stricta exhibited antibacterial activity at a ZOI of 8 mm against K. pneumoniae and 14 mm against S. aureus, whereas Coelogyne cristata showed an inhibition of about 12 mm against S. aureus while its MIC was 31.25 mg/mL 34. P. maculata extracts also showed a good ZOI ranging from 7.75-16 mm in diameter and a very low MIC value ranging from 0.875-14.875 mg/ mL. This result indicates that Pleione maculata is as effective as many other medicinal plants and antimicrobial agents. P. maculata and its closely related genus Coelogyne have not been the subjects of existing antibiofilm research reports.

CONClUsiON
Oxidative stress causes imbalances of oxygen species in the body. It results in health problems such as cancer, cardiovascular disease, neurological disorders, liver malfunction, and other complications caused by DNA damage. Coelogyne and its related genus, Pleione, have previously been studied for their antioxidant and antibacterial properties. However, there were no antibiofilm investigations reported thus far. Pleione and its members, on the other hand, remain an unknown orchid genus. There is a lack of documentation of its antibiofilm efficacy and quantifiable TPC, TFC, or antioxidant activity. As a result, this study aimed to determine the bioactivity of Pleione maculata. P. maculata has potent antioxidant and antibiofilm properties attributed to secondary metabolites such as phenolics and flavonoids. Antibiotic-resistant pathogens forming biofilms are a global concern. Primarily due to the treatment failures for infectious diseases. Pleione maculata exhibits antibacterial and antibiofilm action against Staphylococcus aureus, Klebsiella pneumoniae, and Proteus mirabilis. Future research can focus on developing unique and broad-spectrum antimicrobial compounds. Further studies are required to understand how the extracts work. This study demonstrates the therapeutic potential of Pleione maculata. It establishes the importance of this epiphyte in pharmaceutical and nutraceutical companies. Besides, creating awareness for preventing its extinction and overexploitation.