UHPLC,ATR-FTIR analysis of Nymphoides indica rhizome extract and determination of antioxidant & antibiofilm potential

Abstract

The aim of the study was to perform comprehensive phytochemical analysis, UHPLC, ATR-FTIR profiling and evaluation of various biological activities of N. Indica rhizome. The phytochemical analysis indicated presence of alkaloids, saponins, and triterpenes. In the antioxidant assay, the methanolic (IC₅₀ 40.3±0.04) and chloroform fractions (IC₅₀ 40.05±0.21) showed highest DPPH inhition. Like wise, methanolic fraction showed highest FRAP value (756.2±0.06) followed by chloroform (225.0±0.04) and ethyl acetate fractions (193.0±0.21). In the antimicrobial assays, chloroform (MIC < 0.156 mg/ml) and methanol fractions (MIC 0.625 mg/ml) fractions showed significant inhibition of Klebsiella pneumoniae. Also a significant antibiofilm of biofilm formation was recorded by chloroform (IC₅₀ 1.73 mg/ml) and ethyl acetate fractions (IC₅₀ 1.76 mg/ml). It was concluded that the N. Indica rhizome posess antioxidant, antimicrobial and antibiofilm potential, that may be attributed to high flavonoid contents.

Keywords

ayurveda biofilm root extracts antioxidant rhizome

1 Introduction

The aquatic plants from wetlands are not only considered as essential part of the aquatic ecosystem but also serve as important source of food, construction material and medicine [1]. To date, more than 100 vascular aquatic plants families have been reported that has unique structural diversity [2], and has valuable folklore applications in traditional medicine.

In Pakistan, wetlands are mainly located near delta of various rivers including country’s largest river “River Indus”. Amongst various aquatic medicinal plants, the genus Nymphoides is quiet diverse with 50 species that differ based on biotic properties [2, 3]. The species including Nymphoides indica Nymphoides indica (L.) Kuntze, Nymphoides peltatum (S.G. Gmel.) and Nymphoides cristata (Roxb.) have been reported so far from various districts of Pakistan including Dera Ismail Khan, Attock, Kashmir and Thatta [4]. The Nymphoides indica (L.) Kuntze (Menyanthaceae) is a rhizome containing perennial plant that has free floating leaves [3]. The plant is commonly consumed as a vegetable by various tribes in subcontinent, and various compounds including seco iridoids, flavonoids, terpenes have previously been isolated from various plant parts (Fig. 1) that are reported to posess antibacterial, antidiabetic, antileishmanial, anticancer and anticonvulsant [5, 6] properties. Based on medicinal importance, we investigated the antioxidant, antibacterial and antibiofilm properties of rhizome extracts of N. Indica.

Fig. 1

Major constituents of N. Indica.

2 Experimental section

2.1 Plant collection and processing

The plant was collected from the wetland of river Indus (District D.I. Khan, KPK, Pakistan) and the herbarium sheets were prepared and submitted to Islamabad herbarium, Quaid I Azam University Islamabad, Pakistan, where voucher specimen were deposited (5298-GA, Accession No. ISL-120019). The rhizomes of collected plant material were separated from rest of the plant and exposed to drying using lab Oven (less than 40°C). After complete drying the roots, were powdered using a lab grinder. Later, the plant material was processed for cold maceration (three times, 10 days each) using Methanol: Water (90:10). Finally, the collected plant extract was dried for solvent evaporation by using rotary evaporator. The dried extract was subjected to liquid-liquid extraction (n-hexane, chloroform and ethyl acetate) [5], to have fractions of classified polarity. Each fraction was freeze dried and stored at –20°C till further use.

2.2 Thin layer chromatography (TLC) profiling

After fractionation, all dried fractions were analysed on silica based TLC cards (F254) using different solvent systems based on polarity (Methanol: chloroform: one drop of ammonia). After development of chromatogram, visualization was performed by using UV lamp (254 and 360 nm) and universal reagents (p-anisaldehyde, vanillin reagent).

2.3 High performance liquid chromatography (HPLC) Profiling

The HPLC profiling for extracts and fractions was performed using UHPLC system (Perken Elmer). The UHPLC system enclosed a degasser, quaternary pump, UV detector, sample collection area (autosampler). A gradient solvent system (acetonitrile: water) was employed for 50 min. All analysis were carried out on C₁₈ column (5μm, 4.6×250 mm, analytical column) (Perkin Elmer, USA).

2.4 ATR-FTIR analysis

Attenuated total reflectance (ATR) FTIR spectrophotometric (Perkin Elmer USA) analysis was performed for identification of various functional groups. Each spectrum was examined at range of 4000–400 cm^–1 at 4 cm^–1 intervals of spectral resolution (average 12 scans). The experiment was performed in triplicate and data was averaged. The acquired data was further checked for literature review and finally interpretation was made using standard tables.

2.5 Phytochemical profiling

The phytochemical investigations on the crude extracts were performed for Glycosides [7], Tannins and Saponins [8], Alkaloids [9] and Terpenes [10].

2.6 Estimation of total phenolics (TPC)

The total phenolic content (TPC) of N. indica extracts was determined using Folin Ciocalteu’s method. Briefly, an aliquot of 0.1 mL of test material was mixed with 1.25 ml reagent and sited intact for 6 min. Subsequently the Na₂CO₃ (1.25 mL; 6%) was added to this solution and sited intact for further 30 min. The absorbance of solution was determined at 725 nm by using a UV-Vis spectrophotometer (Shimadzu). Finally a calibration curve (gallic acid; 0–200μg/mL) was prepared and used to present results as mg (GAE) gallic acid equivalents [11] (Kalita et al., 2013).

2.7 Estimation of total flavonoids (TFC)

The total flavonoid content (TFC) of N. indica rhizome was evaluated by using Aluminium chloride method. In brief, an aliquot of 0.5 mL of extract (various concentrations) were mixed with aluminium chloride (1 mL; 2%) and one mL solution of sodium acetate (4%). The solution was allowed to stay untouched for 20 min and the absorbance was recorded afterwards at 440 nm using spectrophotometer. By following same practise, a calibration curve was constructed (quercetin; 0–250μg/ mL). The results were stated as mg rutin equivalent (RE)/gm [12].

2.8 Antioxidant analyses

2.8.1 DPPH assay

The DPPH experiment was performed in accordance with modified method [8]. The DPPH solution (0.1 mL; 0.1 mM) was prepared and mixed with 0.1 mL of extract (diverse concentrations). This mixture was incubated at room temperature in a dark place (for 30 min at least). Consequently, the absorbance of mixture was measure at 517 nm by using spectrophotometer. Quercetin was used as standard drug. The inhibition percentage was calculated as; $% inhibition = (1 - Abs of sample / Abs of control \times 100)$

2.8.2 Ferric reducing antioxidant power (FRAP) Assay

In this assay, an improved method [13] was employed. In the first instance, the assay reagent (FRAP reagent) was prepared by mixing TPTZ solution (10 mM, 5 mL), FeCl₃.6H₂O (20 mM,5 mL) and acetate buffer (300 mM,50 mL) at room temperature (25°C). The extracts (70μL) were mixed with FRAP solution (1425μL) and kept intact in dark area for at least 30 min. Later the absorbance was recorded using a spectrophotometer at 593 nm. Trolox standardisation curve (range 0–300μg/mL) was prepared and results were expressed mg trolox equivalent (TE)/g.

2.9 Antimicrobial assays

2.9.1 MIC and MBC

The plant material was evaluated for antimicrobial activities against Staphylococcus aureus (ATCC 33862), Klebsiella pneumoniae (ATCC 1705), E-coli (ATCC 29050) and Pseudomonas aureginosa (ATCC 27853). The 96 microwell plates were loaded with 50μl of the over night-grown bacterial cultures (1.5×10⁷ CFU/mL), followed by addition of 50μl of crude extract (8 dilutions). The plates were incubated at 37°C for 24 hr. Afterwards, 40μl of resazurin (0.015 %) was added to each well followed by incubation at 37°C for further 60 min. The readings were recorded using colorimetric method. For MBC values, bacterial suspension (10μL) from the MIC microwell was relocated to already prepared agar plates (Muller Hinton) and incubated for 24 hrs. Afterwards, bacterial growth on the agar plates was observed. All samples were loaded in triplicate. Ciprofloxacin was used as standard [14].

2.9.2 Antibiofilm assay

The biofilm formation assay was performed using 12 well polystyrene plates with slight modified method. Briefly, bacterial strain (Pseudomonas aureginosa ATCC 27853) was inoculated in LB medium (280μL; turbidity adjusted with 0.5 McFarland standard). Afterwards, an aliquot of 100μL of tested extract (various concentrations) was added to bacterial culture followed by incubation at 37°C for 24 hrs. Afterwards, the cell growths in plates was measured at 620 and 592 nm. For quantification, the biofilms in 12 well plates was stained using crystal violet (0.01%for 30 min) and then 95%ethanol was added in stained cells and absorbance was recorded at 592 nm [15].

The %inhibition will be calculated using following formula $% inhibition = (1 - Abs of sample / Abs of control \times 100)$

3 Results and discussion

The phytochemical analysis indicated presence of saponins, alkaloids and triterpenes. (Table 1). This analysis revealed a slight presence of alkaloids in the ethyl acetate and chloroform fractions. Likewise saponins were present in aqueous fraction, whereas flavonoids were detected in fractions of medium polarity (Methanol, chloroform and ethyl acetate) that may be responsible for biological activities. It is interesting to note here there most recently; a series of flavonoids were isolated from aerial parts with excessive amount of 3,7-Di-O-methylquercetin-4-O-β-glucoside [5]. The fractions were further processed for HPLC profiling. During this profiling (Fig. 2), the methanol, chloroform and ethyl acetate fractions showed intense peaks. Although the chromatograms are complicated, however flavonoids, iridoids and triterpenes are mainly expected as reported previously for leaf fractions [5]. The functional groups identification was performed using ATR-FTIR, that confirmed presence of various functional groups including phenolics, carboxylic acids, alkanes amines, aromatic amines, alkyl halides were present in the extract (Table 2, Fig. 3).

Table 1
Phytochemical screening of the N. indica rhizome extracts

Extract fraction Phytochemical assessment

Flavonoids Steroids/triterpenoids Alkaloids Tannins Saponins

Chloroform ++ + + – –

Ethyl acetate +++ + ++ – –

MeOH 90% ++ ++ – ++ –

n-Hexane – – – – –

Aqueous – – – ++ ++

Extract fraction	Phytochemical assessment
Chloroform	++	+	+	–	–
Ethyl acetate	+++	+	++	–	–
MeOH 90%	++	++	–	++	–
n-Hexane	–	–	–	–	–
Aqueous	–	–	–	++	++

exceedingly present: dark coloration, discreetly present: moderate coloration, slightly present: very little coloration, absent: no change in colour; Phytochemicals: +++ exceedingly present, ++ discreetly present, + slightly present, –absent [16].

Fig. 2

HPLC profile of N. Indica rhizome extracts.

Table 2

ATR-FTIR analysis of N. Indica rhizome extracts

Functional groups	Peak range (cm^–1)	Bond	N. indica Extract
Aliphatic Amine	3300–3500	N-H Stretching
Alcohol/Phenol	3200–3600	O-H stretching	3392.2
Alkanes	2850–2970	C-H stretching	3003.6
	2850–2970	C-H stretching	2920
Carbonyl	1690–1760	C-H stretching
Alkenes	1610–1680	-C = C- Stretching	1662.3
Alkyles
R-C(O)-NH2	1590–1655	-N-H bending	1599.2
R-C(O)-NH-R	1510–1560	N-H bending	1514.5
Alkanes	1340–1470	C-H Bending	1434.5
Ester (aromatic)	1365 + 1395	-CH(CH3)₂ Bending	1398.7
	1250–1310	O = C-O-C stretching	1309.2
Alcohol/Carboxylic acid/Ester/Ether	1180–1360	C-O Stretching	1312.5
Aliphatic amines	1050–1300	C-O Stretching	1013.32
Alcohol/Carboxylic acid/Ester	1050–1300	C-O
Aromatic amine	1020–1250	C-N Stretching	1013.8
Carboxylic	910–950	O-H bending	950.46
Aliphatic bromo	500–600		567.2

Fig. 3

ATR-FTIR spectrum related to N. Indica total extract.

A detailed quantitative determination of phenolics, flavonoids and antioxidant profile was performed. Overall, a significantly high amount of phenolics (162.2±0.25 mg/gGAE ¹) was recored in methnolic part followed by chloroform fraction (110.1±0.24 mg/gGAE ¹) (Table 3). Simmilarly, same fractions showed the presence of sigifant concentrations of flavonoids (60.7±0.34 and 72.4±0.02 mg/gRUE¹ respectively). The flavonoids and phenolics are mainly known for high antioxidant activities [17] as indicated in DPPH assay. The fractions of medium plority presented high antioxidant power (Table 3). During the DPPH assay the highest activity was recorded in methanolic fraction (IC₅₀ 40.3±0.04) and chloroform fractions (IC₅₀ 40.05±0.21). The aqueos part presented higher activity (IC₅₀ 74.4±0.02) comapred to ethyle acetate (IC₅₀ 177.2±0.32), that could be due to tanins (poly phenols) [18].

Table 3

Quantitative determination of phenolics, flavonoids and antioxidant profile

Sample	TPC (mg/gGAE¹)	TFC (mg/gRUE²)	DPPH (IC₅₀μg/mL)	FRAP (μM)
Total extract	85.2±0.11	37.3±0.74	53.2±0.14	720.5±0.23
Ethyl acetate	96.6±0.23	30.3±0.32	177.2±0.32	193.0±0.21
Chloroform	110.1±0.24	72.4±0.02	40.05±0.21	225.0±0.04
Aquous	49.6±0.15	61.1±0.03	74.4±0.02	80.4±0.05
Methanol	162.2±0.25	60.7±0.34	40.3±0.04	756.2±0.06
Reference standard			5.5±0.02³

¹mg equivalents per gram of gallic acid, ²mg equivalents per gram of rutin, ³Quercetin (μM).

Since all extracts presented intersting antioxidant activites, it was cosidered essntial to determine the total antioxidant power. For this, FRAP assay was employed that measures the ferric reducing ability of given samples at low pH, when a ferric- tripyridyltriazine (Fe^III-TPTZ) complex is reduced to the ferrous (Fe ^II) form, an intense blue colour. During this assay, methanolic fraction presenetd highest FRAP value (756.2±0.06μM), followed by chloroform (225.0±0.04μM) and ethyl acetate fractions (193.0±0.21μM). The result are indicative of strong antioxidant potential of N. indica rhizome extracts.

In antimicrobial assays, the MIC of N. indica rhizome extracts was determined using 96 micowell plate against both gram positive and negative bacteria. A significantly high activty was recoreded in case of chloroform fraction (MIC < 0.156 mg/mL) against K. pneumoniae followed by methanol fraction (MIC 0.625 mg/mL) (Table 4). Based on phytochemical investigations (Table 1), phenolics and flavonoids were considered, responsible for antimicrobial activity [18]. Furthermore the aqueous fraction showed interesting activities against E.coli (MIC 2.5 mg/mL) and K. pneumoniae and P. aureginosa (MIC 1.25 mg/mL) (Table 4). Since the aqueous fraction was rich in tannins [Table 1], the antibacterial properties of these extracts could be related to tannins since these compounds has the potential to deactivate adhesion of bacteria, enzymes, and cell envelope transport proteins resulting in transformation of microbial morphology [18]. As expected the n-hexane fraction presented significant inhibition (MIC 0.625 mg/mL) of growth activities towards all microbes that is possibly due to presence of fatty acids [20]. After MIC, extracts and fractions were further processed for MBC determination and interesting results were recorded. Overall, the MBC were two to three fold higher than MIC values towards K. pneumoniae and P. aureginosa. However, the range was one to two fold high compared to MIC in case of S. aureus and E.coli (Table 4).

Table 4

Antibacterial activity of N. Indica rhizome extracts (mg/ml)

Plant extract	MIC/MBC (mg/ml)
	S. aureus		E.coli		K. Pneumoniae		P. aureginosa
	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC
Total extract	2.5	> 10	5	5	2.5	> 10	5	> 10
Chloroform	5	5	1.25	2.5	< 0.156	> 10	1.25	> 10
Ethyl acetate	5	5	1.25	2.5	1.25	> 10	1.25	> 10
n-hexane	2.5	5	1.25	2.5	0.625	> 10	1.25	> 10
Aqueous	5	> 10	2.5	> 10	1.25	> 10	1.25	> 10
Methanol 90%	1.25	2.5	1.25	5	0.625	> 10	2.5	> 10

Crude extracts and fractions were finally processed for determination of antibiofilm activities using a biofilm producer strain Pseudomonas aureginosa (Table 5). The highest inhibition of biofilm was recorded in case of chloroform (IC₅₀ 1.73 mg/mL) and ethyl acetate fractions (IC₅₀ 1.76 mg/mL) respectively. Overall the total inhibition by extract (IC₅₀ 4.8 mg/mL) was considered as moderate (Table 5). Further, a concentration dependant inhibition of biofilm by plants extracts was recorded (Fig. 4). Since both chloroform and ethyl acetate fractions are rich in flavonoids and poly phenols, it was hypothesized that these may contribute towards antibiofilm properties of N. Indica possibly due to overexpression of efflux protein genes [21]. To best of our knowledge, current study is the first to describe antibiofilm properties of N. Indica rhizome extract.

Table 5

Antibiofilm properties of N. Indica rhizome extract

Extract part	Biofilm formation IC₅₀ (mg/mL)
Total extract	4.8
Chloroform	1.73
Ethyl acetate	1.76
n-hexane	2.08
Aqueous	6.16
Methanol 90%	2.11
Standard ¹	0.383

Ciprofloxacin (mg/ml).

Fig. 4

Antibiofilm activities of N.indica fractions at different concentrations.

4 Conclusion

It was concluded that N. Indica rhizome extracts posess moderate antimicrobial, antibiofilm properties.

5 Source of funding

The funding source was SRGP research grant (1402) from Higher education commission (HEC) of Pakistan.

Footnotes

Acknowledgments

The authors acknowledge Higher education commission (HEC) of Pakistan for Research grant award (SRGP 1402) to Dr. Adnan Amin.

Conflict of interest

None.

Author’s contributions

AA and MAK designed and supervised the study plan. MH performed all the experiment. IA and AR performed phytochemical screening. BAK helped in manuscript writing. All authors have read and approved

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Extract fraction	Phytochemical assessment
	Flavonoids	Steroids/triterpenoids	Alkaloids	Tannins	Saponins
Chloroform	++	+	+	–	–
Ethyl acetate	+++	+	++	–	–
MeOH 90%	++	++	–	++	–
n-Hexane	–	–	–	–	–
Aqueous	–	–	–	++	++