Introduction

Plants have been widely used as a source of food, clothing, shelter, fibre, and fodder as well as a remedy to treat chronic diseases since ancient times. The use of ethnomedicinal plants to cure various human and animal diseases has been a part of human tradition and civilization. Traditional plants have potential pharmacological properties because of the abundance of various bioactive compounds [1]. The people living in rural and remote areas are dependent on traditional medicine systems to cure various ailments. About 60% and 80% of the populations of the world and developing countries, respectively, are dependent on ethnomedicines as per the reports of the World Health Organization (WHO) [2]. The interest in the development of medicines from ethnomedicinal plants has increased among researchers due to their large-scale availability, accessibility, and applicability. The traditional medicinal plants are explored for their pharmaceutical properties and measures are taken to integrate them into basic healthcare systems. A course for traditional and complementary medicine (T&CM) released in a publication of the WHO Traditional Medicine Strategy 2014–2023 highlights the requirement for the incorporation of traditional medicine into public health systems [3]. The rise of ineffectiveness, soaring prices, and vast side effects associated with allopathic medicines have shifted the paradigm from the allopathic medicinal system to the traditional medicinal system. It is documented that about 25% of allopathic medicines are obtained from active plant compounds [4]. Furthermore, it is estimated that about 7000 plant-derived medicinal compounds are employed in Western pharmacopoeia and more attention is paid to the preparation of different plant extracts, isolation of bioactive compounds, and evaluation of their different pharmacological properties [5]. The medicinal plants are rich in various secondary metabolites including polyphenols, saponins, amides, alkaloids, carotenoids, flavonoids, terpenoids, glycosides, and tannins with potential biological properties. Therefore, ethnomedicinal plants have been vastly studied for various pharmacological properties including antibacterial, antifungal, antiviral, antimycobacterial, antiprotozoal, antibiofilm, ant-quorum sensing, antioxidant, anticancer, anti-inflammatory, hepatoprotective, anti-antirheumatic, antiulcer, anti-thrombotic, antiatherosclerotic, antisyphilitic, anthelmintic, antiscorbutic, astringent, antiperiodic purgative, antidiarrheal, mosquitocidal, insecticidal, antinociceptive, larvicidal, acaricidal, diuretic and demulcent, antihyperglycemic, antipyretic, emollient, expectorant, analgesic, aphrodisiac, carminative, laxative, and diaphoretic [5, 6].

The plant-derived antioxidants are reported to show significant scavenging of free radicals, reducing cellular damage, age delaying, and preventing aging-associated diseases including cardiovascular, cancer, hepatic, and neurodegenerative [7]. The major categories of free radicals include (a) reactive oxygen species (ROS), (b) reactive sulphur species (RSS), and (c) reactive nitrogen species (RNS), e.g. oxygen singlets, hydroxyl radical, superoxides, hypochlorite, hydrogen peroxide, peroxynitrite radicals, and nitric oxide–induced oxidative stress. The free radicals are reported to give rise to diseases like acute renal failure, cancer, Alzheimer’s disease (AD), Parkinson’s disease, and diabetes. Different traditional medicine systems including Chinese, Unani, and Ayurveda are providing substantial proof regarding the availability and extraction of biologically active and therapeutically important antioxidant compounds from plants [8].

The plant secondary metabolites synthesized in response to environmental stresses and microbial infections possess potent antimicrobial activity against a wide range of microorganisms affecting human health, the food industry, animal husbandry, etc. Besides antimicrobial potential, plant extracts are reported to have antibiofilm and anti-quorum sensing activities against pathogenic microorganisms. Conventional antibiotics are reported less effective against biofilm-forming microbes (> 1000 times more resistant than planktonic form) resulting in enhanced multi-drug resistance and subsequent recurrent infections [9]. Biofilm-forming microbes, their chronic pathogenicity, and enhanced antibiotic resistance are the biomedical concerns demanding efficient antibiofilm alternatives. Biofilm formation is a quorum sensing (QS)–dependent multistep process and plant-derived antibiofilm agents work solely or synergistically against them either by inhibiting their adhesion to surfaces, attenuating the maturation of biofilms, inhibiting QS signalling cascades, etc. [10].

It has been reported that about 65% of people in India use ethnomedicines [11] and out of 17,000 enlisted species of plants in India 7500 have reported medicinal values [4]. In India, plants have been used as a basic medicinal system since Vedic times as the use of plants to cure diseases is mentioned in Rig Veda and Athar Veda [12]. North East India (NEI) includes the states of Assam, Arunachal Pradesh, Mizoram, Nagaland, Sikkim, Meghalaya, Tripura, and Manipur. It encompasses different biomes of the world since it is part of both the Himalayas and Indo-Burma biodiversity hotspots. It constitutes about 50% of India’s biodiversity and is regarded as the richest and most diverse reservoir of plant diversity in India [13].

In this regard, 20 ethnomedicinal plants from different regions of NEI were evaluated for their antioxidant, antibacterial, antibiofilm, and anti-quorum sensing activities. Additionally, the plants were also evaluated for sugars, proteins, β-carotene, vitamin C, total carotenoids, total phenolic content, and total flavonoid content. The plants enlisted in Table 1 were selected based on available traditional knowledge, wide usage, reported biological activities, and availability.

Table 1 Traditional importance, reported biological activities, distribution, habit and habitat, and family of selected ethnomedicinal plants
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Material and Methods

Chemicals and Media

Growth media and other chemicals were purchased from Hi-media. Crystal violet, gentamicin, and other solvents were purchased from Sigma-Aldrich.

Bacterial Strains and Growth Conditions

Gram-positive bacteria, viz. Bacillus cereus, Bacillus subtilis, Listeria monocytogenes, Mycobacterium smegmatis, and Staphylococcus aureus with MTCC 430, MTCC 121, MTCC 839, MTCC 14,468, and MTCC 3160, respectively; Gram-negative bacteria, viz. Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Pseudomonas diminuta, Salmonella typhimurium, and Yersinia enterocolitica with MTCC 40, MTCC 618, MTCC 2297, MTCC 3361, MTCC 3232, and MTCC 859, respectively; and a fungal strain Candida albicans MTCC 183 were purchased from Microbial Type Culture Collection and Gene Bank, Chandigarh, India. An indicator strain of QS Chromobacterium violaceum ATCC12472 and a mutant strain of C. violaceum (CV026) were purchased from American Type Culture Collection (ATCC). Luria–Bertani broth (LB), nutrient broth (NB), and tryptone soya broth (TSB) were used to culture the bacterial strains while potato dextrose broth (PDB) was used for the growth of fungal strain.

Collection of Plants and Preparation of Extracts

Different parts (root, leaf, branch, bark, seed, fruit) of 20 ethnomedicinal plants were collected from different locations in NEI. The plants were identified by the Botanical Survey of India (BSI), Eastern Regional Centre Shillong (reference nos. BSI/ERC/2018/Tech/469 and BSI/ERC/Tech/2020–2021/1491). The collected parts were cleaned, washed, and surface sterilized with 70% alcohol and 5% aqueous sodium hypochlorite solution. After proper washing, 10% (w/v) methanolic extracts were prepared, followed by lyophilization, and storage at − 20 °C [35]. The lyophilized extracts were dissolved in water for performing experiments. The name of the collected plants, extract code name, part used, place of collection, and date of collection are described in the Supplementary Information.

Phytochemical Analysis

Total and Reducing Sugar

Total sugar in the plant powder was estimated as per the method of Dubois et al. [36] The plant extracts (2 mL) were pipetted into a test tube, followed by the addition of 0.5 mL of 80% phenol and 0.5 mL of concentrated sulphuric acid. The intensity of the solution was measured after 30 min of rest at 485 nm using a UV–vis spectrophotometer against distilled water as a blank. Glucose was used as a standard to quantify the content in the unknown sample. Reducing sugar was estimated by the 3,5-dinitrosalicylic acid (DNS) method of Miller et al. [37] where 1 mL of extract was mixed with 80% ethanol and 3 mL of DNS followed by boiling at 90 ℃ for 15 min. The solution was then allowed to cool at room temperature followed by adding 1 mL of 2% sodium potassium tartrate tetrahydrate solution. The intensity of the solution was then measured at 575 nm using a UV–vis spectrophotometer (Cary 60, Agilent UNICO Products and Instruments Inc., Shanghai, China). Fructose was used as a standard.

Vitamin C Content

The AOAC official methods (21st ed.) (2019) (AOAC 967.21) [38] were used for the determination of vitamin C content. The redox titration method using 2,6-dichlorophenol indophenol dye was adopted to assess the vitamin C content of the plant materials. First, indophenol dye (50 mg) was prepared in the distilled water (250 mL) followed by the addition of sodium carbonate (42 mg). Briefly, 2 mL of extract was mixed with 5 mL of metaphosphoric acid followed by titration against indophenol dye until rose pink colour.

β-carotene Content

β-carotene content in plant material was estimated as per the procedure outlined by Biswas et al. [39], where plant powder (1 g) was mixed with 5 mL of chilled methanol followed by centrifugation at 1370 rcf for 10 min. The absorbance of the extract was determined at 449 nm, and concentration was calculated using β-carotene as standard in the range of 0–32 mg mL−1.

Total Carotenoids

Total carotenoid content in the plant material was determined using a spectrophotometer method as described by de Carvalho et al. [40]. One gram of powder was taken and extracted by mixing it in 14 mL hexane and acetone (3:2 v.v−1) solution and using an Eppendorf centrifuge at 10,000 relative centrifugal speed for 10 min at 4 ℃. The obtained supernatants were collected and absorbance was measured at 502 nm using a UV–vis spectrophotometer. The following equation was used to calculate the total carotenoid content in plant powder:

$$\mathrm{Total\; Carotenoids }\left(\frac{\mathrm{mg}}{\mathrm{g}}\right)=\frac{{\mathrm{OD}}_{502} \times 3.12}{\mathrm{mass\; of\; the\; sample }\;(\mathrm{g})} \times 1000$$

Protein Content

Protein content in the plant material was determined according to the procedure outlined by Lowry et al. [41].

Determination of Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)

The methods described by Kara et al. [42] were used for the determination of TPC and TFC of 20 plant extracts. Briefly, a mixture of 25 µL of plant extract (1 mg/mL), 100 µL of 0.075 g/mL sodium carbonate, and 125 µL of 10% v/v Folin–Ciocalteu reagent was incubated for 30 min and absorbance was taken at 765 nm. Total phenolic content was expressed as milligrams of gallic acid equivalent per gram dry weight (mg GAE/g DW) and was measured from the calibration curve of gallic acid (Sigma-Aldrich). For the estimation of TFC, a mixture of 25 µL of plant extract (1 mg/mL), 75 µL of 95% ethanol, 10 µL of 10% w/v potassium acetate, 10 µL of 10% w/v AlCl3, and 140 µL of deionized water was incubated for 40 min and absorbance was measured at 415 nm. Total flavonoid content was expressed as milligrams of quercetin equivalent per gram dry weight (mg QE/g DW) and was calculated from the calibration curve of quercetin (Sigma-Aldrich).

Antioxidant Activity of Plant Extracts

Antioxidant activity of plant extracts was determined by 1,1-diphenyl-2-picryl-hydrazyl assay (DPPH) and reducing power assay mentioned by González-Palma et al. [43].

1-Diphenyl-2-picryl-hydrazyl Assay

Briefly, a mixture of 190 µL of 60 µM DPPH solution and 10 µL of plant extracts (1 mg/mL) was placed in the dark for 30 min. After incubation, absorbance was measured at 517 nm and % DPPH scavenging activity was calculated by using the following formula. Ascorbic acid and quercetin were used as positive controls while methane served as carrier control.

$$\%\;\mathrm{DPPH}\;\mathrm{Scavenging}\;\mathrm{activity}=\left[\left(\frac{OD_{\mathit c\mathit o\mathit n\mathit t\mathit r\mathit o\mathit l}-OD_{\mathit t\mathit e\mathit s\mathit t}}{OD_{\mathit c\mathit o\mathit n\mathit t\mathit r\mathit o\mathit l}}\right)\right]\times100$$

where ODcontrol and ODtest are the absorbances of blank and extracts, respectively.

Reducing Power Assay

Briefly, mixtures of 50 µL plant extract (1 mg/mL), 0.2 mL each of 1% potassium ferricyanide, and 0.2 M phosphate buffer (pH 6.6) were incubated in a water bath at 55 °C for 20 min. After incubation, 250 µL trichloroacetic acid (TCA) was added to the mixture and centrifuged at 1000 rpm for 10 min. Five hundred microliters of supernatant was mixed with 500 µL deionized water and 100 µL of 0.1% FeCl3. The absorbance (OD700) was measured after 10 min which is directly proportional to the reducing power.

Antibacterial Activity

Agar Well Diffusion Assay

To demonstrate the antibacterial activity of different crude extracts, agar well diffusion assay was performed. Briefly, 100 µL of 0.4 OD600 of B. cereus, B. subtilis, L. monocytogenes, M. smegmatis, S. aureus, E. coli, K. pneumonia, P. aeruginosa, P. diminuta, S. typhimurium, Y. enterocolitica, and C. albicans was spread on growth medium agar plates. The plant extract (4 mg/mL), gentamicin (2.5 mg/mL-positive control), nystatin (5 mg/mL-positive control for C. albicans), and methanol (carrier control) were placed into separate wells prepared by metallic borer (6 mm). After an incubation of 24 h at 37 °C, the plates were evaluated for inhibition zones around the wells and diameters of zone of inhibition (ZOI) (mm) were measured [44].

Evaluation of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

MIC and MBC were defined as the minimum concentration of plant extracts where no visible growth was demonstrated and which inhibited the total growth of the bacteria, respectively. Briefly, MIC values were determined by microdilution assay and MBC values were evaluated by placing culture on agar plates from the wells with higher concentrations than MIC values. Subsequently, the plates were incubated at 37 °C for 24 h and bacterial growth was observed after incubation. The concentration at which no growth was observed on the plate was considered the MBC value. The minimum concentration of plant extracts that could inhibit 80% of the bacterial growth was considered to be the MIC80, which was calculated as follows:

$$\mathrm I\mathrm n\mathrm h\mathrm i\mathrm b\mathrm i\mathrm t\mathrm i\mathrm o\mathrm n\;\%=\left[\left(OD_{\mathit c\mathit o\mathit n\mathit t\mathit r\mathit o\mathit l}-OD_{\mathit t\mathit e\mathit s\mathit t}\right)/OD_{\mathit c\mathit o\mathit n\mathit t\mathit r\mathit o\mathit l}\right]\times100$$

where ODcontrol and ODtest are OD600 nm to demonstrate bacterial growth in the positive control (media + culture) and treated samples (media + culture + extract).

All the subsequent experiments were carried out at sub-MICs and the MIC80 and MBC values were estimated only against P. aeruginosa and C. violaceum. Furthermore, the total antibacterial activity (TAA) (mL/g) of plant extracts was calculated from the ratio of the mass of crude extract (mg) obtained from 1 g of plant sample and MIC (mg/mL) [45].

Total antibacterial activity (mL/g) = mass of crude extract per gram of plant/MIC.

Determination of Antibiofilm Activity by Crystal Violet Staining Assay

Effect of Plant Extracts on the Initial Attachment of P. aeruginosa

The antibiofilm activity of the extracts was evaluated against P. aeruginosa by the method described by Famuyide et al. [45] with slight modifications.

Scanning Electron Microscopy

To demonstrate the biofilm inhibitory effect of plant extracts at a microscopic level, SEM was performed. Briefly, P. aeruginosa was grown on coverslips in the presence (treated) and absence (control) of plant extracts. After incubation, the cells were fixed by 2.5% (v/v) glutaraldehyde followed by dehydration using 50, 70, 75, 80, 85, 90, and 95% ethanol and were analysed under SEM (JEOL JSM-6390LV, Tokyo, Japan) [46].

Effect of Plant Extracts on Preformed Biofilm of P. aeruginosa — Biofilm Eradication Assay

To demonstrate the effect of plant extracts on preformed biofilm, the crystal violet staining method mentioned by Famuyide et al. [45] was used. Briefly, biofilm eradication activity of those plant extracts was estimated which exhibited potential biofilm inhibition activity (inhibition of initial attachment) against P. aeruginosa. Biofilms were allowed to form in a 96-well tissue culture plate by incubating 10 µL aliquot of 0.4 OD600 of P. aeruginosa and 190 µL of TSB for 24 h and 48 h at 37 °C in a static condition. After respective incubation periods, potential plant extracts were added at a final concentration of 1 mg/mL (sub-MIC) and were incubated for 24 h at 37 °C. % biofilm eradication was determined by the crystal violet method as mentioned earlier. Methanol and furanone (1 mM) served as the negative and positive control, respectively. The following formula was used to calculate the percentage of biofilm eradication

$$\%\;\mathrm{Biofilm}\;\mathrm{eradication}=\frac{OD_{\mathit c\mathit o\mathit n\mathit t\mathit r\mathit o\mathit l}-OD_{\mathit t\mathit e\mathit s\mathit t}}{OD_{\mathit c\mathit o\mathit n\mathit t\mathit r\mathit o\mathit l}}\times100$$

where ODcontrol and ODtest are the absorbances of untreated control and treated cultures, respectively.

Effect of Plant Extracts on Pyocyanin Production

The method described by Das et al. [47] was used for the quantification of pyocyanin production. The following formula was used to calculate the percentage of pyocyanin inhibition;

$$\%\;\mathrm{Pyocyanin}\;\mathrm{inhibition}=\frac{OD_{\mathit c\mathit o\mathit n\mathit t\mathit r\mathit o\mathit l}-OD_{\mathit t\mathit e\mathit s\mathit t}}{OD_{\mathit c\mathit o\mathit n\mathit t\mathit r\mathit o\mathit l}}\times100$$

where ODcontrol and ODtest are the absorbances of untreated control and treated cultures, respectively.

Additionally, to validate the inhibition of pyocyanin by plant extracts was not due to their bactericidal nature, the bacterial growth of the treated and untreated samples was recorded at 600 nm after 24 h of incubation.

Effect of Plant Extracts on Quorum Sensing of C. violaceum

Violacein Quantification Assay

The violacein quantification method described by Moradi et al. [48] was used for the estimation of violacein inhibition. The following formula was used to calculate the percentage of violacein inhibition;

$$\%\;\mathrm{Violacein}\;\mathrm{inhibition}=\left[\left(\frac{OD_{\mathit c\mathit o\mathit n\mathit t\mathit r\mathit o\mathit l}-OD_{\mathit t\mathit e\mathit s\mathit t}}{OD_{\mathit c\mathit o\mathit n\mathit t\mathit r\mathit o\mathit l}}\right)\right]\times100$$

where ODcontrol and ODtest are the absorbances of untreated and treated cultures, respectively.

Additionally, to demonstrate that violacein inhibition was due to anti-QS effects and not due to the bactericidal effect of extracts, log10CFU/mL value of all the treated and untreated samples was counted after 24 h of incubation.

Evaluation of Anti-quorum Sensing Activity Through Quorum Sensing Signal Inhibition

To determine the violacein inhibition of plant extracts via interruption of QS signalling, a biosensor strain C. violaceum CV026 was used in addition to C. violaceum CV12472. CV026 is incapable of producing violacein due to a mutation in the acyl-homoserine lactone (AHL)-producing gene. To prove that violacein inhibition activity of plant extracts is due to their interruption in QS signalling, CV12472 was grown in the presence (1 mg/mL) of plant extracts supplemented with and without external AHL, viz. hexonyl homoserine lactone (C6-AHL) at a concentration of 0.225 µg/mL. Similarly, CV026 was allowed to grow in the presence and absence of C6-AHL. The experiment was set up as discussed above and % violacein production was calculated [48].

Statistical Analysis

All experiments were performed in triplicate, and the obtained results are expressed as the mean values and standard deviations. Statistical difference was evaluated by using Student’s t-test (two-tailed) for unpaired samples. Statistics were calculated in GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA), in which a p-value of ≤ 0.01 was considered statistically significant.

Results

Phytochemical Analysis

Total and Reducing Sugar

Sugar functions as a food as well as a central signalling or regulatory molecule in plants, modulating gene expression in areas such as growth, development, metabolism, stress response, and disease resistance. The total sugar content ranged from 2.92 to 30.6 mM with the highest amount in D. indica at 30.6 mM, followed by A. hookeri at 28.7 mM, and the lowest was in D. esculentum at 2.92 mM (Table 2).

Table 2 Phytochemical analysis of the selected ethnomedicinal plants
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The reducing sugar of the studied plant samples ranged from 0.44 to 14.5 mM (Table 2). Among the twenty plant samples studied, reducing sugar content was found highest in A. sativum at 14.5 mM, followed by D. indica at 12.5 mM, and lowest in D. esculentum at 0.44 mM, followed by O. corniculata at 0.45 mM. In similar studies, Preetha et al. [49], Ayam et al. [50], and Choudhury et al. [51] reported that the reducing sugar of A. sessilis, A. hookeri, and D. esculentum was 56.1 ± 0.20 (mg g−1), 0.18 ± 0.01 mg 100 g−1, and 0.13 ± 0.01 mg g−1 dwt, respectively.

Vitamin C

Vitamin C is an important redox buffer and cofactor for enzymes that regulate photosynthesis, hormone production, and the regeneration of other antioxidants. From Table 2, it is evident that P. peepuloides has the highest amount of vitamin C content of 31.3 mg g−1, followed by A. sessilis (29.9 mg g−1), while the lowest vitamin C content was found in A. sativum 8.41 mg g−1. In a similar study, Preetha et al. [49] found 1.25 µg g−1 vitamin C content in A. sessilis. The vitamin C content of A. hookeri in our study was found to be comparable to that obtained in an investigation by Ayam et al. [50]. Our study demonstrated the vitamin C concentration of D. esculentum is 19.6 mg g−1, which is lower than the 46.0 mg g−1 reported in research by Choudhury et al. (2017). The vitamin C content of most of the plants examined in this study has never been studied before to the best of our knowledge.

β-carotene

β-carotene aids in normal vision, morphogenesis, cell proliferation, and differentiation and is important for iron utilization and humoral immunity. Plants contain β-carotene, a pigment that gives them their colour. The β-carotene concentration in Table 2 was found to be between 0.29 and 8.91 mg g−1. The highest concentration of β-carotene was found in D. indica (8.91 mg g−1), followed by A. hookeri (8.09 mg g−1). A. sativum has the lowest β-carotene concentration (0.29 mg g−1), followed by G. lanceifolia (0.77 mg g−1). In a study carried out by Devi et al. [52], the β-carotene content of A. hookeri was found to be 0.60 mg 100 g−1.

Total Carotenoids

Carotenoids are required for photosynthesis and photoprotection in plants. They are important light-harvesting pigments and photosystem structural components. Total carotenoids in plant materials ranged from 14.9 to 267 mg g−1 in the current study (Table 2). C. sinensis has the highest carotenoid concentration (267 mg g−1), followed by M. micrantha with 229 mg g−1. A. sativum, on the other hand, had the lowest concentration of 14.9 mg g−1. Carotenoids have the antioxidant feature of being very powerful physical and chemical quenchers of singlet oxygen (1O2) as well as potent scavengers of other reactive oxygen species (ROS). The presence of carotenoids in plant extracts has been shown to protect against a variety of ROS-mediated diseases, including cardiovascular disease, cancer, neurological disorders, and photosensitive or eye-related ailments [53].

Protein Content

Proteins are essential biomolecules required for the normal function, structural maintenance, and regulation of the body of all living organisms. Protein concentrations among the tested extracts ranged from 5.65 to 283 mg g−1 (Table 2). D. indica has the highest protein content at 283 mg g−1, followed by A. hookeri at 170 mg g−1. A. paniculate had the lowest concentration of 5.65 mg g−1, followed by C. pallida Aiton with 5.69 mg g−1. C. pallida seeds were found to have about 22% protein as reported by Ukil et al. [54]. Antimicrobial, antioxidant, ribosome-inactivating, and neuro-modulatory properties have been found in proteins isolated from plants.

TPC and TFC

The Folin–Ciocalteu method was employed for the determination of TPC in the plant extracts. The values were evaluated from the gallic acid standard curve with R2 = 0.95 and were expressed as mg GAE/g DW. The amount of the TPC in the plant extracts was found as CSMD (31.0), APMF (30.6), AHMF (28.6), PPMF (22.7), MMMF (19.2), DIMD (19.2), EKMD (13.9), JGMD (13.9), CPMF (10.1), ASMD (9.66), GAMD (9.37), HCMF (7.74), GLMD (6.74), DEMD (6.61), ERMD (6.32), ASMF (5.97), IAMF (5.97), SDMF (5.97), ODMF (5.55), and OCMF (5.32) mg GAE/g DW (Table 2).

Similarly, the TFC values were evaluated from the quercetin standard curve with R2 = 0.99 and were expressed as mg QE/g DW. The values ranged between 1.74 and 68.2 mg QE/g DW with highest the TFC of CSMD (68.2) followed by GAMD (63.9) while the lowest TFC was reported in DIMD (1.74) mg QE/g DW (Table 2).

Antioxidant Activity of Plant Extracts

The scavenging effect of plant extracts on DPPH radical shown in figure demonstrates the following order: CSMD (87.5%) > APMF (83.9%) > HCMF (83.0%) > MMMF (82.2%) > AHMF (81.2%) > PPMF (80.2%) > GAMD (77.5%) > GLMD (75.7%) > ASMD (75.7%) > CPMF (74.8%) > ODMF (74.2%) > OCMF (72.0%) > SDMF (66.6%) > DEMD (61.0%) > ERMD (52.6%) > JGMD (53.3%) > EKMD (52.6%) > ASMF (51.8%) > IAMF (47.7%) > DIMD (25.8%). The positive controls quercetin and ascorbic acid demonstrated % DPPH scavenging of 87.4 and 92.0, respectively (Fig. 1).

Fig. 1
figure 1

Evaluation of the antioxidant activity of the extracts by DPPH assay. The tested plant extracts at a concentration of 1 mg/mL demonstrated significant % DPPH scavenging activity in comparison to positive controls quercetin and ascorbic acid. The highest and lowest % DPPH scavenging activity of 87.59 and 25.78% was exhibited by C. sinensis (CSMD) and D. indica (DIMD), respectively. Methane (carrier control) demonstrated insignificant (5.5%) % DPPH scavenging activity. One milligram per millilitre in the X-axis depicts the tested concentration of plant extracts as well as the positive controls. Error bars represent the standard deviations of three measurements

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Figure 2 depicts the reducing powers of tested plant extracts at a concentration of 1 mg/mL. The decreasing order of the reducing power was CSMD > APMF > PPMF > OCMF > AHMF > MMMF > SDMF > ERMD = GLMD > GAMD > HCMF = ODMF > EKMD = CPMF > ASMD > IAMF > ASMF = JGMD > DIMD > DEMD. CSMD demonstrated a maximum reducing power of 2.9, followed by APMF (2.85), PPMF (2.80), and OCMF (2.55) while the least reducing power was exhibited by DEMD (0.15).

Fig. 2
figure 2

Evaluation of the antioxidant activity of the extracts by reducing power assay. The extracts showed variable absorbance at 700 nm in the range of 2.9–0.15 which is directly proportional to the reducing powers. The highest and lowest absorbances (reducing powers) of 2.9 and 0.15 were exhibited by C. sinensis (CSMD) and D. esculentum (DEMD), respectively. One milligram per millilitre in the X-axis depicts the tested concentration of plant extracts. Error bars represent the standard deviations of three measurements

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Evaluation of Antibacterial Activity and MIC, MBC Values of Plant Extracts

Of the twenty plants, seven demonstrated antibacterial activity against the tested pathogens (Supplementary Information). The ZOI against Gram-positive bacteria ranged between 11 and 23 mm with a maximum ZOI of 23 mm by OCMF against S. aureus and a minimum ZOI of 11 mm by SDMF against M. smegmatis. Similarly, ZOI against Gram-negative bacteria ranged between 10 and 22 mm with maximum and minimum ZOIs of 22 and 11 mm by OCMF and GLMD against P. aeruginosa and S. typhimurium, respectively. The antibacterial results (ZOIs) demonstrated that BC, BS, EC, KP, LM, MS, PA, PD, ST, SA, and YE are highly susceptible to OCMF, ODMF and SDMF, OCMF, OCMF, GAMD, OCMF, OCMF, OCMF, OCMF, OCMF, and APMF (larger ZOI) and least susceptible to SDMF, APMF, SDMF, SDMF, GLMD and SDMF, SDMF, SDMF, APMF and GLMD, GLMD, GLMD, and SDMF, respectively (smaller ZOI). Furthermore, no antibacterial activity was exhibited by AHMF, ASMF, ASMD, CPMF, DIMD, DEMD, EKMD, ERMD, HCMF, IAMF, JGMD, MMMD, and PPMF against any of the tested strains. Also, methanol (M) does not show any antibacterial activity while gentamicin showed prominent activity against all the tested bacteria. Furthermore, none of the extracts inhibited C. albicans while nystatin demonstrated potential antifungal activity (Table 3).

Table 3 Diameter of zone of inhibition (mm) of plant extracts against pathogenic microorganisms
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The MIC80 and MBC values of the active extracts respectively ranged between 1.2–1.9 mg/mL and 1.6–2.3 mg/mL against P. aeruginosa and 1.1–1.7 mg/mL and 1.3–2.1 mg/mL against C. violaceum. The MIC values also demonstrated that P. aeruginosa and C. violaceum are more susceptible to OCMF and ODMF, respectively, and least susceptible to SDMF. Furthermore, TAA values were also evaluated as described earlier which demonstrated that GLMD and SDMF have the highest (274.61 mL/g) and lowest (150.0 mL/g) TAA against P. aeruginosa, respectively, while GLMD and GAMD demonstrated the highest (274.61 mL/g) and lowest (153.57 mL/g) TAA against C. violaceum, respectively (Table 4).

Table 4 MIC80 (mg/mL), MBC (mg/mL), and total antibacterial activity(mL/g) of active plant extracts against P. aeruginosa and C. violaceum
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Demonstration of Antibiofilm Activity of active plant extracts Against P. aeruginosa

Effect of Plant Extracts on Inhibition of Biofilm Formation (Initial Attachment)

The result demonstrated the inhibition of biofilm formation against P. aeruginosa by plant extracts in the range of 8.99–88.3%. In reference to the established criteria, the percentage of biofilm inhibition greater than 50% is considered good antibiofilm activity while % inhibition in the range of 0–49% is considered poor or weak. In this context, 9 extracts demonstrated good antibiofilm activity, viz. CSMD (88.3%) > ODMF (77.1%) > GAMD (76.6%) > APMF (75.9%) > MMMF (74.9%) > SDMF (67.3%) > GLMD (67.1%) > OCMF (63.8%) > JGMD (56.3%). The extracts significantly inhibited the initial attachment or adherence of cells to the surfaces in comparison to the untreated control. However, no inhibition was seen in methanol while furanone (positive control) showed 94.85% inhibition (Fig. 3).

Fig. 3
figure 3

Demonstration of the antibiofilm effect of the active extracts against P. aeruginosa. The tissue culture plate method demonstrated significant inhibition (≥ 50%) of initial attachment of P. aeruginosa to TCP by the extracts (CSMD, ODMF, GAMD, APMF, MMMF, SDMF, GLMD, OCMF, JGMD) at sub-MIC in comparison to untreated control. Furanone (F) and methanol (M) are positive and negative controls, respectively. One milligram per millilitre in the X-axis depicts the tested concentration of plant extracts while furanone (F) and methanol (M) were used at 1 mM and 10% v/v, respectively. Error bars represent the standard deviations of three measurements. ***, p < 0.01 compared with the control

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Scanning Electron Microscopy

The SEM images also confirmed the inhibition of biofilm by the plant extracts. It is evident from Fig. 4 that a matrix of densely packed P. aeruginosa cells along with extracellular polymeric substances (EPS) network could be seen in absence of extract (control); however, a significant reduction in cell density attached to the surfaces as well as EPS production could be seen in treated samples.

Fig. 4
figure 4

Scanning electron microscopy. The figure demonstrates inhibition of bacterial attachment to the surface in presence of different plant extracts, viz. CSMD, ODMF, GAMD, APMF, MMMF, SDMF, GLMD, OCMF, and JGMD; a network of cells embedded within matrix could be seen in control; scale bar of 5 µm and magnification 3500 × 

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Effect of Plant Extracts on Preformed Biofilm

The plant extracts that attenuated the initial attachment of bacteria were evaluated for their activity on preformed biofilm (24 h and 48 h). Six plant extracts, viz. CSMD, ODMF, GAMD, APMF, SDMF, GLMD, and JGMD reduced biofilm biomass of P. aeruginosa at 24 h post-development with percentage inhibition of 84.2%, 66.1%, 72.0%, 58.9%, 60.6%, 61.4%, and 54.2%, respectively. Similarly, the effect of plant extracts on biofilm eradication or biofilm maturation on 48-h preformed biofilm demonstrated significant results in CSMD (73.0%), GAMD (61.9%), SDMF (53.9%), and GLMD (60.9%) (Fig. 5).

Fig. 5
figure 5

Evaluation of the antibiofilm effect on preformed biofilm of P. aeruginosa. The graph demonstrates the eradication and inhibition of biofilm maturation of biofilm (≥ 50%) by the extracts CSMD, ODMF GAMD, APMF, SDMF, GLMD, and JGMD in 24-h period of preformed biofilm. Similarly, CSMD, GAMD, SDMF, and GLMD eradicated or inhibited biofilm maturation (≥ 50%) in 48-h period of preformed biofilm. Error bars represent the standard deviations of three measurements

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Pyocyanin Activity

The quantification of the pyocyanin assay demonstrated significant pyocyanin reduction against P. aeruginosa by some plant extracts. Of the 20 plant extracts, 10 demonstrated significant (≥ 50%) pyocyanin inhibition, viz. GAMD (83.9%), APMF (83.6%), SDMF (83.5%), ODMF (75.8%), DIMD (74.1%), GLMD (73.7%), CSMD (65.9%), MMMF (65.0%), OCMF (60.0%), and CPMF (53.5%). The lowest pyocyanin inhibition was reported in AHMF (0.72%) followed by IAMF (4.16%) and DEMD (7.00%). Methanol showed insignificant pyocyanin inhibition of 3% while furanone demonstrated 88.0% inhibition. Furthermore, a uniform bacterial growth was observed in all the treated (OD600 = 1.95–2.10) and untreated samples (OD600 = 2.15) validating that pyocyanin inhibition was due to the inhibitory effect of the extracts on virulence production and not due to their toxicity (Fig. 6).

Fig. 6
figure 6

Antipyocyanin activity of the extracts. The figure depicts significant (≥ 50%) pyocyanin inhibition in P. aeruginosa by some plant extracts, viz. GAMD, APMF, SDMF, ODMF, DIMD, GLMD, CSMD, MMMF, OCMF, and CPMF. The figure also demonstrated no significant growth change (absorbance 600) of P. aeruginosa in treated samples in comparison to untreated control. Furanone (F) and methanol (M) are positive and negative controls, respectively. One milligram per millilitre in the X-axis depicts the tested concentration of plant extracts while furanone (F) and methanol (M) were used at 1 mM and 10% v/v, respectively. Error bars represent the standard deviations of three measurements. ***, p < 0.01 compared with the control

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Anti-quorum Sensing Activity

Violacein Inhibition Assay

Figure 7 demonstrates the inhibition of violacein production by the tested plant extracts. The result demonstrated the inhibition of violacein production in the range of 88.8–2.93%. The maximum violacein inhibition was reported in CSMD (88.8%) followed by APMF (81.8%) while the lowest activity was reported in IAMF (2.93%) followed by PPMF (12.9%). Methanol and furanone demonstrated violacein inhibition of 1.0% and 94.8%, respectively. Furthermore, almost similar log10CFU/mL values of treated (6.5–6.9log10CFU/mL) and untreated (6.9log10CFU/mL) samples after 24 h incubation demonstrated that the violacein inhibition was due to anti-QS effects of the extracts and not because of their bactericidal activity.

Fig. 7
figure 7

Effect of the extracts on violacein production. A strong violacein inhibitory activity was shown by some plant extracts (≥ 50%) against C. violaceum. The figure also illustrates no significant change in log10CFU/mL of C. violaceum in treated and untreated samples. Furanone (F) and methanol (M) are positive and negative controls, respectively. One milligram per millilitre in the X-axis depicts the tested concentration of plant extracts while furanone (F) and methanol (M) were used at 1 mM and 10% v/v, respectively. Error bars represent the standard deviations of three measurements. ***, p < 0.01 compared with the control

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Evaluation of Anti-quorum Sensing Activity Through Quorum Sensing Signal Inhibition

We proposed that the antibiofilm activity of plant extracts could be because of the interruption of QS. The plant extracts that exhibited both antibiofilm and ant-QS activities were evaluated for violacein production by CV12472 in presence of external C6-AHL. It was found that violacein production was restored in CV12472 treated with plant samples after adding C6-AHL signifying that the antibiofilm activity of the plant extracts could be due to their interruption of QS signalling. CV026 used as a positive control also produced purple pigment after the addition of C6-AHL while no violacein production was observed in C6-AHL-deficient CV026. The violacein production was restored up to 90.8% (Fig. 8).

Fig. 8
figure 8

Anti-violacein activity of the extracts via quorum sensing inhibition. The figure shows restoration of violacein production in active extract-treated C. violaceum (CV12472) supplemented with exogenous acyl homoserine lactone, viz. C6-AHL. AHL-deficient strain CV026 also restored violacein synthesis in presence of C6-AHL. PE + AHL: plant extract supplemented with AHL. Error bars represent the standard deviations of three measurements

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Discussion

Plants are significantly contributing to basic healthcare despite the advancements in conventional modern medicines. Natural products, predominantly from plant sources, are referred to as the important source of novel and potent therapeutics. Plants are a reservoir of different bioactive compounds like phenolics, polyphenolics, and flavonoids. Therefore, they are being explored for the discovery of novel compounds with different biological activities including antimicrobial, antibiofilm, and antioxidant properties [46]. The plant-derived antioxidants including ascorbic acid, polyphenols, gallic acid, and carotenoids were reported to decrease oxidative stress in humans through scavenging, reduction of lipid peroxidation, and nitrosation reactions [8]. The ethnomedicinal plants from NEI are being used in the folk medicinal system to treat diseases including fever, asthma, skin allergies, diabetes, menstrual cramps, diarrhoea, cough, and hypertension [55]. In this context, the current study was carried out to evaluate the antioxidant properties of different plant extracts from NEI by DPPH and reducing power assays. The purple-coloured DPPH solutions turn colourless due to the scavenging of DPPH by antioxidants or free radicals. The higher the change in colour intensity, the higher is the free radical scavenging activity and thus, the higher the antioxidant potential of the tested extract. Various tested plant extracts demonstrated significant antioxidant activity via DPPH assay. Among all C. sinensis var. assamica, A. paniculate, A. hookeri, P. peepuloides, M. micrantha, and H. cordata demonstrated the highest DPPH scavenging activity as a high degree of colour change of the DPPH solution (decrease in absorbance) was observed in them. Similarly, reducing power assay also demonstrated the significant antioxidant of the tested extracts particularly C. sinensis var. assamica, A. paniculate, O. corniculate, P. peepuloides, A. hookeri, and M. micrantha since the highest colour shift (yellow to green) was observed among them. The assay is based on the reduction of Fe3+/ferricyanide complex to the ferrous form in presence of reductants. It has been reported that the reducing properties demonstrated antioxidant potential by breaking the free radical chain by the donation of hydrogen atoms [56].

Food and nutrition science has developed an interest in phenolic and flavonoid-rich diets owing to their different biological activities. Furthermore, phenolic-rich plant materials are widely used in the food industry since they enhance the nutritional value and quality of food by reducing the oxidative degradation of lipids. Phenolics act as strong antioxidants either by donating electrons or by enhancing the endogenous synthesis of antioxidant molecules within the cell [57]. In the current study, the TPC values ranged between 31.0 and 5.32 mg GAE/g DW. The highest TPC values of CSMD (31.0), APMF (30.6), AHMF (28.6), PPMF (22.7), and MMMF (19.2) could be attributed to their strongest antioxidant potential values discussed earlier. Similarly, TFC values ranged between 68.2 and 1.74 mg QE/g DW and the high TFC of CSMD, GAMD, APMF, AHMF, and MMMF could be responsible for their strong antioxidant activity. A similar study carried out by Chavan et al. [58] demonstrated TFC and TPC values in the range of 0.1–15.3 mg of rutin equivalents/gram fresh weight and 0.3–28.5 mg of tannic acid equivalents/gram fresh weight, respectively, for different Ceropegia sp. The other phytochemical analyses including the determination of vitamin C, β-carotene, total carotenoids, total sugars, reducing sugars, and protein content of all the plants were carried out. The analysis demonstrated their abundance in the plant samples signifying the dietary and pharmaceutical values of the extracts.

Plants continue to be one of the leading sources of therapeutic compounds including antimicrobials against pathogenic microorganisms, viz. bacteria, fungi, viruses, etc. to minimize the spread and recurrence of infectious diseases. The use of ethnomedicinal plants to impede the spread of infectious diseases offers the most emergent non-toxic and eco-friendly antimicrobial approach [59]. Our results demonstrated that C. sinensis var. assamica, A. paniculate, G. acuminata, G. lanceifolia, O. corniculate, O. debilis, and S. dulcis exhibited potent antibacterial activity against the tested pathogenic strains. The extracts exhibited inhibitory effects against both Gram-positive and Gram-negative bacteria. The tested strains are important in the human healthcare system, food industry, and animal husbandry since they are associated with clinical infections, food spoilage, and food-borne diseases. Subsequently, the MIC and MBC values of active extracts against P. aeruginosa and C. violaceum showed their variable susceptibility towards them. It was found that P. aeruginosa was more susceptible to O. corniculate and least susceptible to S. dulcis while C. violaceum was most susceptible to O. debilis and least susceptible to S. dulcis. The TAA value gives the volume of solvent to which 1 g of the extract is added and still shows antimicrobial activity. For example, the highest TAA 274.61 (mL/g) of G. lanceifolia against P. aeruginosa reported in the current study signifies that the extract will show antibacterial activity against it even if 1 g of the extract is dissolved in about 0.27 L of the solvent. Furthermore, MIC and TAA values are important pharmacological tools for determining the potency (mg/mL) for isolating bioactive compound and efficacy (mL/g) of the extract (plant selection). A similar study carried out by Elisha et al. [59] demonstrated the antibacterial activity, MIC, MBC, and TAA values of nine plant extracts, viz. Cremaspora trifloral, Maesa lanceolata, Maesa lanceolata, Hypericum roeperianum, Elaeodendron croceum, Calpurnia aurea, Hypericum roeperianum, Heteromorpha arborescens, and Pittosporum viridiflorum against both Gram-positive and Gram-negative bacteria.

Microbial antibiotic drug resistance and biofilm induction are the biggest challenges the healthcare sector is prevailing through. The microbes develop biofilms in response to environmental cues to develop resistance against conventional chemotherapies (antibiotics) [60]. P. aeruginosa, a ubiquitous opportunistic pathogen infecting animals, humans, insects, and plants, forms strong biofilms and is responsible for 57% of nosocomial infections [46]. The plant extracts are reported to exhibit antibiofilm activity against a wide range of microbes. The current study demonstrated a significant antibiofilm activity of some extracts against P. aeruginosa by inhibiting initial attachment to the surface. Furthermore, the plant extracts that inhibited ≥ 50% biofilm were checked for their effect on preformed biofilm. Of all the active extracts, C. sinensis var. assamica, G. acuminata, S. dulcis, and G. lanceifolia significantly eradicated the biofilm of 24- and 48-h periods. The antibiofilm activity of the extracts could be due to the effect on cellular hydrophobicity, inhibition of EPS secretion, cell-associated virulence factors, overall interruption of the biofilm-related signalling cascade, etc. P. aeruginosa is associated with the production of virulence factors, viz. pyocyanin, alginate, rhamnolipid, elastase, and protease which induce pathogenicity to it and help invasion to host tissues. The secretion of these virulence factors is regulated by cell-to-cell communication called QS. Pyocyanin is a blue-coloured phenazine compound that produces ROS and induces cellular damage by attacking host biological membranes and exhausting intracellular c-AMP as well as ATP levels, thus impairing cellular respiration as well [61]. The study demonstrated significant pyocyanin inhibition by some plant extracts at sub-MIC. G. acuminata, S. dulcis, A. paniculate, etc. demonstrated the highest reduction in green colour intensity, thus indicating the highest pyocyanin reduction.

The abundance of different phytochemicals in traditional plants including phenolics, flavonoids, terpenoids, polystyrenes, quinones, and alkaloids has been reported involved in QS inhibition and attenuation of microbial pathogenesis associated with their biofilms [62]. In this regard, some of our tested extracts demonstrated significant violacein inhibition against CV12472 at sub-MIC. The results indicate the presence of quorum sensing inhibitors (QSIs) in the plant extracts. Furthermore, plant secondary metabolites especially flavanones, quinones, terpenoids, and phenolics are reported to interrupt QS signalling or damage QS receptors owing to their similar chemical structure to that of AHLs (QS signals). In an attempt to justify that our active plant extracts inhibited biofilm and QS by inhibiting AHL production via interruption of QS signalling, external AHL was added to extract-treated CV12472. The results demonstrated resumption of violacein synthesis in extract-treated CV12472 which indicated that the extracts inhibited AHL synthesis by interrupting QS. To validate our results that the reduction of pyocyanin and violacein production was due to interruption of QS and not due to cytotoxicity or bactericidal nature of plant extracts, log10 CFU/mL and growth pattern (OD600) of all the treated and untreated samples remained the same after 24 h of incubation. The results demonstrated consistency with the findings of Moradi et al. [48].

Various QSIs have been discovered from different sources including microorganisms, plants, and chemical synthesis which play a vital role in impeding QS-regulated biofilm formation and production of virulence factors. Different mechanisms have been established regarding the inhibition of microbial communication by QSIs, viz. competitive binding of QSIs at QS signal molecule binding sites, inhibition of synthesis of autoinducers (AIs), degradation of signal molecules, immunological scavenging of signal molecules, interfering QS signalling cascades and impeding QS-regulated genes, etc. [63,64,65,66].

Conclusion and Future Perspectives

The discovery of plant-derived novel antioxidants and antimicrobial agents offers an effective, non-toxic, and sustainable alternative to conventional drugs. In this regard, the ethnomedicinal plants of NEI were evaluated for their phytochemical composition, antioxidant, antibacterial, antibiofilm, and anti-QS activities to validate their traditional importance. Our study demonstrated the presence of variable concentrations of different phytochemicals like phenolics, flavonoids, total carotenoids, β-carotene, vitamin C, sugars, and proteins in the selected extracts. The abundance of these phytochemicals in the extracts attributed significant antioxidant potential to them. Some of the plant extracts examined in the study demonstrated inhibitory potential against both sessile and planktonic forms of bacteria, therefore could be potential candidates for isolation of antimicrobial and antibiofilm compounds. In conclusion, this study provides comprehensive detail of the biological activities of the plants of NEI used in traditional medicine systems and provides validation for their inclusion in the basic healthcare system. The active plant extracts in this study could be developed as strong antioxidant and antimicrobial agents subjected to further investigation and validation. Furthermore, it is imperative to determine the mechanism of action of the plant extracts on different pathogenic microorganisms.