Phytochemical analysis, in vitro antioxidant and antimicrobial activities of male flower of Juglans regia L.

ABSTRACT

The male flower of Juglans regia L., were investigated for its in vitro antioxidant activity, antimicrobial activity, and chemical constituents. The antioxidant activity showed that the methanol extract of J. regia male flower (MEJR) had highest scavenging potential than the other solvents (ethanolic = EEJR and aqueous = AEJR). The antimicrobial activity showed that Staphylococcus aureus and Escherichia coli were the most sensitive organisms and significant activity was also recorded against both the fungal strains tested, with highest activity against Candida albicans. Totally, 26 constituents were identified by high-resolution-liquid chromatography-mass spectrometry analyses from which seven compounds were identified first time from the extract.

KEYWORDS:

• Antimicrobial
• Antioxidant
• FTIR
• Juglans regia L
• LCMS

Introduction

The Juglandaceae family includes several genera among which Juglans genus is the important representative, with 7–45 species. Among these Juglans regia L. is one of the premium tree traditionally cultivated for its valuable wood and fruits. The seed is a nut of high economic interest to the food industry and is globally popular and valued for its nutritional, health, and sensory attributes.^[1] The vast biodiversity of Himalaya provides this royal species mostly in the Kashmir region, growing up to 25–35 m.

Juglans regia is considered to treat a variety of health complaints traditionally, including Cancer, Inflammation, Diabetes, Antiradicalar, Hyperhidrosis, Antidiarriec, Prostate, Antiradicalar, and Cardiovascular disorders.^[2–5] However, researchers investigated that almost all parts of the plant are important against different health disorders as well as for preservation of food grains.^[6] The extracts from J. regia nut inhibited oxidative damages,^[7–10] inflammation,^[11,12] tumor growth,^[8,13] antiwrinkle, and photoageing.^[14] Kernels as a dietary food, against diabetes, hypoxia, some skin diseases, and inflammation^[15,16]; leaves as antidiarrheals, anthelmintic, depurative^[17] and also mixed with stored-grains as an insecticide and fungicide.^[17,18] Stem bark as an astringent, anthelmintic, depurative, bactericide, diuretic, digestive, laxative, stimulant, detergent, and insecticidal.^[19] Juglans regia L. shell is reported for polishing gun-casings, jewelry, and metal material and is used as media to separate water and crude oil.^[20]

Juglans regia L. is a good source of flavonoids, Polyphenols, flavonols, carbohydrates, fatty acids, cardiac glycosides, steroids, minerals, tannins, protein, dietary fiber, melatonin, plant sterols, α‐tocopherol, folate, tannins, vitamin A and C, and vitamin E family compound.^[1,21–23] Several studies demonstrated the antimicrobial activity of phenolic extracts,^[20–25] making them as best substitute to antibiotics and food preservatives. Juglans regia L. is a natural product of high economic interest to the food industry and is very popular and largely consumed as royal food globally and valued for its nutritional, health, and sensory attributes.^[1] There is an extended interest in using natural antimicrobial compounds, due to the increasing resistance to antibiotics.^[24] Although all parts of this valuable plant has been investigated for different biological properties, no study regarding the phytochemical analysis, antioxidant and antimicrobial activity has been reported yet for the male flower of J. regia L. from Himalayan region. The objective of this study is thus to evaluate the phytochemical analysis, antioxidant, and antimicrobial activity of male flower of J. regia L. and its further utilization in food products.

Materials and methods

Reagents and instruments

2,4,6-tris(2-pyridyl)-1,3,5-triazine (TPTZ), α,α-Diphenyl-2-picrylhydrazyl (DPPH), 2,2′- azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ascorbic acid (ABA), sodium nitrite, aluminum chloride, gallic acid, and quercitin were purchased from Sigma-Aldrich Chemical Co.(St. Louis, Mo., USA). All organic solvents were of analytical grade (Merck, Darmstadt, Germany), a Multimode reader (Tecan, Austria) were used for the determination of antioxidant and antimicrobial activities. Spectrum RX1 FT-IR spectrophotometer (Perkin Elmer, USA) was used for FT-IR spectra recording and liquid chromatography/mass spectrometry (UHPLC-PDA-Detector ­Mass spectrometer (HR-LCMS 1290 Infinity UHPLC System, 1260 infinity Nano HPLC with Chipcube, 6550 iFunnel Q­TOFs), Agilent Technologies, USA) were used for polyphenolics analysis and identification.

Collection and preparation of plant extracts

The male flower part of J. regia L. (locally referred to as Doon Fidin) were collected from the Indian Himalayan region of Jammu and Kashmir during the flowering stage (April 2014), at an altitude 33.7167°N 74.8333°E. The plant materials were identified by Dr. D. Kumarasamy, the designated plant taxonomist of the Department of Botany, Annamalai University, with herbarium voucher no. ABH-2023. The plant material was shade dried and powdered by using electric grinder. Plant material was soaked into solvents (95% methanol and ethanol and water) in a ratio of 1: 6 (w/v) for 48 h at 20°C with vigorous shaking. The extract was subjected to evaporation under vacuumed pressure and residual material was considered as source of crude extract and was stored at 4ºC for further analysis. The extraction yield has been calculated by fallowing the method of^[25] directly by using the formula

$\mathrm{E}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{y}\mathrm{i}\mathrm{e}\mathrm{l}\mathrm{d}(\mathrm{\%})=\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{r}\mathrm{u}\mathrm{d}\mathrm{e}\mathrm{e}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}/\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{o}\mathrm{r}\mathrm{i}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{l}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{x}100$

Antioxidant assays

ABTS radical scavenging assay

ABTS method measures the capacity of different compounds to scavenge the 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid radical cation (ABTS•+).^[26] The antioxidant activity was measured in a reaction mixture containing 0.5 mL of 15 µM H₂O₂, 0.5 mL of 7 mM ABTS, and 50 mM sodium phosphate buffer, pH 7.5. The absorbances were recorded by spectrophotometer at 734 nm and were compared with standard ascorbic acid. IC₅₀ value is the concentration of sample required to inhibit 50% of ABTS•+ production. The percentage of ABTS radical scavenging was calculated as given below:

$\mathrm{\%}\mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\left[\mathrm{A}\mathrm{B}\mathrm{T}{\mathrm{S}}^{\bullet +}\right]=\frac{\mathrm{A}0-\mathrm{A}1}{\mathrm{A}0\mathrm{x}}\times 100$

Where A0 was the absorbance of the control and A1 was the absorbance in the presence of the sample of ascorbic acid (ABA).

Ferric reducing antioxidant power (FRAP) assay

The FRAP assay was performed using a TPTZ solution.^[27] The working solutions were prepared by mixing 25 mL of acetate buffer (pH 3.6, 300 mM), 2.5 mL of TPTZ solution (10 mM), and 2.5 mL of FeCl 3 · 6H2O solution (20 mM), and were kept at 37°C. The sample solutions with different concentrations (mg/mL) were then mixed with the working solutions and were incubated under darkness at 37°C for 30 min. The absorbance was measured at 650 nm. The antioxidant activities were expressed as µmol/L FeSO4 equivalent/mg extracts, called FRAP values. A higher FRAP value corresponds to a greater antioxidant activity.

DPPH radical scavenging capacity

Free radical scavenging property of different extracts of J. regia male flower against DPPH• (2, 2-diphenyl-1-picrylhydrazyl) was determined spectrophotometrically by the method of.^[28] DPPH• is a stable free radical, becomes a stable diamagnetic molecule when gets reduced. DPPH• donate hydrogen when reacts with an antioxidant and gets reduced, this change in color was measured (from deep violet to light yellow) which is directly proportional to the amount and nature of radical scavenger present.

Antimicrobial activity

Microorganisms

The antimicrobial activity of male flower of J. regia was tested against two Gram positive bacterial strains viz. Staphylococcus aureus and Bacillus subtilis, two Gram negative bacterial strains viz. Escherichia coli and Proteus vulgaris, and against two fungal strains viz. Candida albicans and Candida glaborata. The microorganisms were obtained from Rajah Muthiah Medical College and Hospital, Annamalai University, India. The stock cultures were maintained on Muller–Hinton Agar (for bacteria) and Sabouraud dextrose agar medium (for fungi) at 4°C.

Disc diffusion assay and determination of minimum inhibitory concentration (MIC)

Antimicrobial activity of different extracts of male flower of J. regia was determined by using disc diffusion method of^[29] with little modifications. The susceptibility tests were performed on Muller–Hinton Agar (bacteria) and Sabouraud Dextrose Agar (fungi). 50, 25, and 12.5 mg (diluted with mg/mL 5% dimethyl sulfoxide (DMSO)) of all extracts were impregnated on the filter paper discs (6 mm) and used for the study. Ciprofloxacin (5 mg/disc) for bacteria and Amphotericin B (20 mg/disc) for fungi were used as positive reference standards to determine the sensitivity of the tested strains and 5% DMSO was used as blind control. Finally, the inoculated plates were incubated at 37°C for 24 h(for bacteria), 28°C for 48 h (for Candida) and the inhibition zones were observed including the diameter of the disc (6 mm).

MIC of the male flower of J. regia was tested by the twofold serial dilution method of.^[30] The extracts were dissolved in 5% DMSO to obtain 100 mg/mL stock solution. 0.5 mL of stock solution was incorporated into 0.5 mL of Mueller Hinton broth (bacteria) to get the concentration of 100 mg/mL and serially diluted to achieve 50, 25, and 12.5 mg/mL. Fifty microliters of standardized suspension of the test organism was transferred on to each tube. The control tube contained only organisms, without extracts and 5%DMSO was used as blind control. The culture tubes were incubated at 37°C for 24 h. The lowest concentrations, which did not show any growth of tested organisms after macroscopic evaluation was determined as MIC.

Phytochemical investigation

Total phenolic content (TPC)

TPC of MEJR, EEJR, and AEJR were analyzed by using Folin–Ciocalteu method fallowed by^[31] with little modifications. 0.5 mL of extracts (0.5 mg/mL) were diluted with 6 mL of double-distilled water, then mixed with 0.5 mL of 1N Folin–Ciocalteu’s reagent and were allowed to settle for 5 min. consequently 2 mL of 20% Na₂CO₃ solution were supplemented. Absorbance was measured at 750 nm by using UV–VIS spectrophotometer (Tecan, Austria), after the incubation of 1 h at RT. Gallic acid has been used as standard. Total content of phenolics were expressed as mg gallic acid equivalents/100g DW.

Total flavonoids content (TFC)

TFC of MEJR, EEJR, and AEJR were determined on the basis of formation of flavonoid-aluminum complex by using spectrophotometric method of^[32] with little modifications. 0.5 mL of 2% AlCl3 (aluminum chloride) with methanol were added to 0.5 mL of extract. Absorbances were measured at 430 nm after the incubation of 15 min at room temperature. Quercitin has been used as standard. The total content of flavonoids was expressed as mg quercitin equivalents/100 g DW. The values were presented as means of triplicate analyses.

Fourier transform infrared (FT-IR) spectroscopy analysis

FT-IR spectra were recorded by using Spectrum RX1 FT-IR spectrophotometer (Perkin Elmer, USA) working in 4000–400 cm⁻¹ region, Outfitted with a KBr beam splitter, DTGS detector and Nichrome source. Total of 100 scans were acquired for Final spectrum with 4 cm⁻¹ resolution. For the preparation of translucent sample discs, the dried powdered extract was encapsulated in KBr. pellet.

High resolution-liquid chromatography-mass spectrometry analysis (HR-LCMS)

The HR-LCMS analysis of MEJR was analyzed by with some modifications, by using UHPLC-PDA-Detector ­Mass spectrometer (HR-LCMS 1290 Infinity UHPLC System, 1260 Infinity Nano HPLC with Chipcube, 6550 iFunnel Q­TOFs), Agilent Technologies, USA. For chromatographic separation, an Agilent 1200 Series HPLC system (Agilent Technologies, USA) equipped with a binary gradient solvent pump, HiP Sampler, column oven and MS Q-TOF with Dual AJS ES Ion Source. Samples were separated on SB-C18 column (2.1 × 50 mm, 1.8-particle size; Agilent Technologies, USA) maintained at 25°C. The solvents used were: water containing 0.1% HCOOH and methanol containing 0.1% HCOOH. The following gradient elution program at a flow rate of 0.4 mL min⁻¹ was applied. MS detection was performed in MS Q-TOF Mass spectrometer (Agilent Technologies). The identified constituents were quantified on the basis of their peak areas and comparison with a calibration curve obtained with the corresponding standards. Linearity ranges for calibration curves were specified.

Statistical analysis

All of the examinations were executed in triplicate, and the values were expressed as the mean ± standard deviation. The results were evaluated through analysis of variance (ANOVA) with Duncan’s multiple range tests (p < 0.05) using SPSS.

Results and discussion

Antioxidant activity of JR

To evaluate the antioxidant activities of three different extracts of male flower of J. regia L., DPPH, ABTS, and FRAP assay were analyzed as shown in Table 1. The DPPH, ABTS, and FRAP assays are based on electron transfer between sample and the reagent radical and are measured by evaluating their color changes spectrophotometrically. All three extracts of J. regia showed the significant free radical scavenging activity against DPPH, BTS, and FRAP assay, comparing with the high antioxidant effect of ascorbic acid. However, MEJR showed the highest antioxidant activity having IC₅₀ value of 66.80 ± 2.13 for DPPH, 53.95 ± 6.46 for ABTS, and also FRAP value of 43.60 ± 4.83 µmol/L FeSO4/mg extract as compared with EEJR having IC₅₀ value of 75.17 ± 4.43for DPPH, 63.40 ± 5.73 for ABTS and FRAP value of 54.35 ± 3.12 µmol/L FeSO4/mg extract, followed by AEJR having IC₅₀ value of 76.76 ± 3.75 for DPPH, 64.30 ± 7.23 for ABTS and FRAP value of 52.70 ± 2.90 µmol/L FeSO4/mg extract. The free radical scavenging activity of different extracts are comparable with previously reported studies on leaves, bark, stem, and nuts of J. regia by using DPPH, ABTS, and FRAP assays.^{[8,22,33–36]}

Table 1. Antioxidant activity of different extracts of male flower of J. regia L.

		IC50(µg/ml)			Contents (mg/g DM)
Sample	Yield (%)	DPPH^•	ABTS^•+	FRAP (µmol/L FeSO4/mg extract)	TFC	TPC
MEJR	12.3	66.80 ± 2.13^b	53.95 ± 6.46^b	43.60 ± 4.83^b	144.62 ± 2.40^c	129.76 ± 3.11^c
EEJR	8.8	75.17 ± 4.43^c	63.40 ± 5.73^c	54.35 ± 3.12^c	137.81 ± 3.28^b	124.12 ± 2.45^b
AEJR	7.9	76.76 ± 3.75^c	64.30 ± 7.23^c	52.70 ± 2.90^c	131.79 ± 4.69^a	122.1 ± 2.80^a
ABA	——–	51.68 ± 1.10^a	41.17 ± 1.79^a	33.20 ± 0.57^a	—————	——————

DPPH radical scavenging activities; ABTS radical scavenging activities; Ferric reducing antioxidant power (FRAP) and Total flavonoid contents (TFC) and Total phenolic contents (TPC) of male flower of J. regia L. Samples (µmol/L FeSO4 equivalent/mg extracts). Data are the mean of three independent analyses of each representative variety (mean ± SD; n = 3).

^{a, b, c} Data are shown as mean ± SD, n = 3. Data with different super script letters in the same column are significantly different (p < 0.05).

The results of the total phenolic content evaluated using Folin-Ciocalteu method, are shown in Table 1 to support the antioxidant activity of the male flower of J. regia. MEJR showed the higher TPC (129.76 ± 3.11 mg/g DM) as compared to EEJR (124.12 ± 2.45 mg/g DM) and AEJR (122.1 ± 2.80 mg/g DM), which is similar with previously reported TPC (116.39 ± 5.63 and 92 ± 1.40 mg GAE/g) of J. regia nuts in methanolic and petroleum ether extracts, respectively.^[8]

Flavonoids are secondary metabolites, with several health benefits such as antioxidant, anti-inflammatory, and antimicrobial activities.^[37] Rutin, quercetin, Gallic acid, and kaempferol are major flavonoids reported previously in J. regia.^[38,39] The TFC values in the present study were highest in MEJR (144.62 ± 2.40 mg/g) followed by EEJR (137.81 ± 3.28 mg/g) and AEJR (131.79 ± 4.69 mg/g), which is significant than previously reported TFC in the methanolic and ethyl acetate extract of Shell of J. regia where TFC was 48.90 ± 0.7 and 80.40 ± 0.55 mg QEs/g extract, respectively.^[40]

Antimicrobial activity

The antimicrobial potential of male flower of J. regia against two Gram positive and Gram negative bacteria and two fungi were evaluated by using disc diffusion method and determination of minimum inhibitory concentration and the results are presented in Table 2. The results revealed that the male flower of J. regia showed significant antimicrobial activity against all the bacterial and fungal strains tested and Gram positive bacteria were more susceptible than Gram negative bacteria and fungi. The mean zone of inhibition produced by all the extracts ranged from 9.28 ± 0.5 to 46.13 ± 4.1 mm and the MIC value were between 2.6 and 4.7 mg/mL. The MEJR showed highest antimicrobial activity with the highest mean zone of inhibition (46.13 ± 4.1 mm) and lowest MIC (2.6 mg/mL) values against S. aureus followed by EEJR against E. coli (39.19 ± 4.3 mm; MIC = 3.9) and AEJR against B. subtilis (39.15 ± 2.9 mm; MIC = 3.0). MEJR also showed the highest mean zone of inhibition against E. coli (44.7 ± 3.2 mm; MIC = 3.4;) and P. vilgaris (43.12 ± 1.8 mm; MIC = 3.0 µg/mL). The highest antifungal activity was also observed in MEJR against C. albicans (40.57 ± 3.7 mm) and C. globarata (39.4 ± 2.5) followed by EEJR against C. albicans (33.73 ± 3.52 mm) and AJR against (36.2 ± 1.8).

Table 2. Antimicrobial activity of different extracts of male flower of J. regia L.

	Mean zone of inhibition (mm)^x,y
	MEJR			EEJR			AEJR			Control drug		MIC (mg/mL)
Name of the organism	MEJR (12.5mg/mL)	MEJR (25mg/mL)	MEJR (50mg/mL)	EEJR (12.5mg/mL)	EEJR (25mg/mL)	EEJR (50mg/mL)	AEJR (12.5mg/mL)	AEJR (25mg/mL)	AJR (50mg/mL)	Ciprofloxacin (5mg/mL)	Amphotreicin-B 20mg/mL	MEJR	EEJR	AEJR
Gram positive bacteria
Staphylococcus aureus	15.62 ± 1.32	29.50 ± 2.01	46.13 ± 4.21	11.43 ± 0.70	19.35 ± 1.35	34.73 ± 1.95	10.29 ± 1.10	17.05 ± 2.45	36.26 ± 4.18	41.1 ± 3.75	NT	2.6^a	4.5^b	4.5^b
Bacillus subtilis	13.28 ± 2.15	24.3 ± 1.35	42.2 ± 2.56	10.9 ± 1.03	21.56 ± 3.4	33.8 ± 2.22	7.46 ± 0.7	18.6 ± 3.1	39.15 ± 2.9	44.3 ± 2.53	NT	3.0^a	5.3^c	4.7^b
Gram negative bacteria
Escherichia coli	19.35 ± 2.00	31.5 ± 1.4	44.7 ± 3.2	14.2 ± 1.3	25.75 ± 2.4	39.19 ± 4.3	11.3 ± 0.16	22.6 ± 1.8	38.4 ± 3.2	42.6 ± 3.82	NT	3.4^a	3.9^b	4.7^b
Proteus vulgaris	16.17 ± 1.1	29.79 ± 3.6	43.12 ± 1.8	11.3 ± 0.8	21.2 ± 3.5	37.69 ± 2.1	9.28 ± 1.3	17.4 ± 2.7	37.3 ± 2.19	40.24 ± 3.5	NT	3.0^a	4.0^b	4.0^b
Fungi
Candida albicans	18.3 ± 2.7	27.2 ± 1.4	40.57 ± 3.7	12.65 ± 1.8	24.16 ± 1.31	37.73 ± 3.52	7.62 ± 0.6	17.8 ± 3.25	31.7 ± 3.7	NT	37.14 ± 2.18	2.5^a	3.0^a	3.0^a
Candida glabrata	15.2 ± 1.9	26.8 ± 3.6	39.4 ± 2.5	10.3 ± 0.9	24.12 ± 0.96	33 .9 ± 1.23	9.4 ± 1.4	19. 35 ± 2.1	36.2 ± 1.8	NT	39.21 ± 4.74	3.0^a	4.0^b	4.0^b

MIC: minimum inhibitory concentration; NT: not tested. (X = Mean zone of three assays. Y = Inhibition zones including the diameter of the disc (6 mm)).

^{a, b, c} Data are shown as mean ± SD, n = 3. Data with different super script letters in the same column are significantly different (p < 0.05).

Recently, Farooqui et al.^[41] found the bark of J. regia against 15 bacteria’s (viz, Staphylococcus aureus, Streptococcus pyogenes, Enterobacter cloacae, Citrobacter freuendii, etc.) with MIC ranging from 0.31 to >5 (mg/mL). Zakavi et al. (2013)^[42] reported that the ethanolic and aqueous extracts of J. regia bark against some oral Bacteria (Staphylococcus aureus, Streptococcus sanguis, Streptococcus salivarius, and Streptococcus mutans), having MIC up to 5 mg/mL. Cruz-Vega et al. (2008)^[43] also found leaves and bark as the active aerial part of J. regia against microbes with MICs ranging from 100 to 125 μg/mL. Noumi et al. (2010)^[44] reported the antibacterial effect of bark of J. regia L. with MIC ratio ranging from 0.006 to 3.125 (mg/mL). The differences registered between our results and previously reported data could be attributed to the extraction procedure, the plant origin, the tested microorganisms, and the size of the inoculums. The results of antifungal activity of different extracts of J. regia L., is comparable with the results of Pereira et al., 2007,^[45] Oliveira et al., 2007,^[46] and Sytykiewicz et al., 2015^[47] screened leaves and green husks of J. regia from Portugal and Poland, against Candida spp. with significant activity.

FT-IR spectral analysis

FT-IR spectral analysis of MEJR revealed the occurrence of various functional groups in it. Spectral data confirmed the existence of bioactive functional groups like alkanes (C–H stretch, C–H rock), alkenes (–C = C– stretch, = C–H bend), alkynes (–C ≡ C–H: C–H stretch), 1°, 2° amines and amides (N–H stretch), α, β–unsaturated aldehydes and ketones (C = O stretch), 1° amines (N–H bend), aromatics (C–C in–ring), aliphatic amines (C-N stretch), and alkyl halides (C–Cl stretch). IR absorption frequencies and the representative spectra are shown in Table 3 and Fig. 1, respectively. The analyzed functional groups give the probable identification of compounds present in the extract and support the data analyzed by HR-LCMS.

Table 3. Major bands observed in the FT-IR spectra of MEJR.

	IR νmax (cm^−1) (Vibration mode)
S.no	Frequency(m^–1)	Bond	Functional groups	Probable Phytochemical
1	720.22	C–H rock, C–Cl stretch, C–H oop, N–H wag	Alkanes, alkyl halides, aromatics, 1°, 2° amines	Alkaloids, flavonoids, tannins, poly phenols, carboxylic acid etc., containing phytochemicals
2	831.74	C–Cl stretch, C–H oop, N–H wag, = C–H bend	alkyl halides, aromatics, 1°, 2° amines, alkenes
3	910.19	N–H wag, = C–H bend, O–H bend	aromatics, alkenes, carboxylic acids
4	1057.79	C–N stretch	aliphatic amines
5	1164.65	C–H wag(–CH2X), C–O stretch, C–N stretch	alkyl halides, alcohols, carboxylic acids, esters, ethers, aromatic amines
6	1197.18	C–H wag(–CH2X), C–O stretch, C–N stretch	alkyl halides, alcohols, carboxylic acids, esters, ethers, aromatic amines
7	1244.85	C–N stretch, C–H wag (–CH2X), C–O stretch	aromatic amines, alkyl halides, alcohols, carboxylic acids, esters, ethers,
8	1277.53	C–N stretch, C–H wag (–CH2X), C–O stretch	aromatic amines, alkyl halides, alcohols, carboxylic acids, esters, ethers,
9	1377.33	C–H bend	alkanes
10	1409.55	C–C stretch (in–ring)	Aromatics
11	1463.71	C–H bend, C–C stretch (in–ring)	Alkanes, aromatics
12	1604.70	N–H bend	1° amines
13	1650.62	N–H bend, –C = C– stretch	1° amines, alkenes
14	1709.51	C = O stretch,	α, β–unsaturated aldehydes, ketones
15	2852.41	C–H stretch, O–H stretch	Alkanes, carboxylic acids
16	2922.15	C–H stretch, O–H stretch	Alkanes, carboxylic acids
17	2957.39	C–H stretch, O–H stretch	Alkanes, carboxylic acids
18	3010.03	=C–H stretch, C–H stretch	Alkenes, aromatics
19	3331.81	N–H stretch, O–H stretch (H–bonded)	1°, 2° amines, amides, alcohols, phenols

Figure 1. FTIR spectrum of MEJR.

Phytochemical composition of MEJR

The methanolic extract of J. regia male flower (MEJR) were selected for phytochemical analysis by using HR-LCMS, on the basis of significant antioxidant, extraction yield (%), TPC and TFC content, and antimicrobial activity. With the retention times, absorbance spectra and MS data, chemical composition of MEJR possess 26 bioactive compounds Fig. 2. The chemical formulae, retention time, and mass of the compounds are listed in Table 4. Some of the above compounds have already been identified in different parts of J. regia L. from the different ecological conditions like Arginine in nuts of J. regia from New Zealand,^[38] Docosahexaynoic acid in nuts from six J. regia Cultivars grown in Portugal,^[48] 11-amino-undecanoic acid (ursolic acid) were reported in green husks of J. regia in China,^[49] 2,2-Dimethyl-3-Oxo-Butyric Acid 2-Trimethylsilanyl; Quercetin-3-O-glucuronide; Hexanoic acid, trimethylsilyl ester; Oleic acid; n-Hexadecanoic acid and 1,2–Benzene dicarboxylic acid, bis-(2-methyl propyl) ester were reported in leaves, green husk, stem bark, and nuts of J. regia.^{[50,51,10,52]} Chemical constituents like, Elephantopin, Gamma-L-Glutamyl-cysteine, Artemisinin, Madecassic acid, Dihydromyricetin, Swietenine, Securinine, and Sphinganine were reported first time from the J. regia as per our best knowledge. The differences in the phytochemistry may be varying by season, habitat, or the ecological conditions of plants.

Table 4. Identification of phenolic acids and flavonoids in the male flower of J. regia L. by high-resolution liquid-chromatography mass spectrometry (HR-LCMS) analysis.

Rt (min)	Mass (m/z)	Identification	Molecular formula	Molecular weight (g/mol)
4.98	126.0156	Unknown
5.89	174.1094	Arginine	C6 H14 N4 O2	174.20
7.149	316.1474	4,7,10,13,16,19- Docosahexaynoic acid (omega-3 fatty acid)	C22 H32 O2	328.496
7.977	360.1231	Elephantopin	C19 H20 O7	360.362
8.399	250.0636	gamma-L-Glutamyl-Lcysteine	C8 H14 N2 O5 S	250.269
9.79	282.142	Artemisinin	C15 H22 O5	282.332
9.795	201.1706	11-amino-undecanoic acid (ursolic acid)	C11 H23 N O2	201.31
10.046	228.1448	Pro-Ile	C11 H20 N2 O3	228.292
10.817	138.0671	Unknown
11.26	173.1058	Unknown
11.522	185.1419	Unknown
11.839	245.1352	Gly-Ala-Val	C10 H19 N3 O4	245.279
12.475	219.1084	Pantothenic Acid (vitamin B5)	C9 H17 N O5	219.24
13.096	136.0134	3,4,5-trihydroxy benzoic acid (Gallic acid)	C7 H6 O5	170.12
13.608	161.083	Indole-3-ethanol	C10 H11 N O	161.20
14	610.0622	Quercetin-3-O-rutinoside	C27H30O16	610.52
14.591	217.1105	Securinine	C13 H15 N O2	217.268
14.686	172.1164	Decanoic acid	C10H20O2	172.27
15.024	240.195	Unknown
15.354	504.3487	Madecassic Acid	C30 H48 O6	504.708
15.854	320.0514	Dihydromyricetin	C15 H12 O8	320.25
16.411	280.342	(9Z,12Z)-9,12-Octadecadienoic acid (Linoleic acid)	C18H32O2	280.45
16.601	320.0512	Dihydromyricetin (Ampelopsin)	C15 H12 O8	320.25
18.012	273.2655	C16 Sphinganine	C16 H35 N O2	273.469
18.765	315.2768	Unknown
19.041	478.12	Quercetin-3-O-glucuronide (Miquelianin)	C21H18O13	478.36
19.227	460.2321	Gln-Trp-Lys	C22 H32 N6 O5	460.535
19.667	282.2231	(9Z)-Octadecenoic acid (Oleic acid)	C18H34O2	282.47
20.507	304.2999	Unknown
20.789	363.3137	Unknown
21.354	568.273	Swietenine	C32 H40 O9	568.661
21.976	256.321	n-Hexadecanoic acid (Palmitic acid)	C14H28O2	256.43
22.035	278.235	1,2 – Benzene dicarboxylic acid, bis (2-methyl propyl) ester	C16H22O4	278.3435
22.593	276.1869	4-oxo-9Z,11Z,13E,15E-octa-decatetraenoic acid (unsaturated fatty acids in plants)	C18 H28 O2	276.40
24.89	393.3705	Unknown

Rt: Retention time

Figure 2. HR-LCMS spectrum of MEJR.

Conclusion

The results obtained from the study showed that male flower of J. regia possess significant antioxidant and antimicrobial activity and can be used as an easily accessible source of natural bioactive compounds. However, tested strains were more susceptible to MEJR as compared to EEJR and AEJR and significant free radical scavenging activity was also observed in MEJR. Seven new bioactive compounds were also identified from the MEJR along with other known compounds. Thus, the study explored the fact that J. regia L. have great reservoir of new antioxidant and antimicrobial agents and hence demonstrated that J. regia L. is a potential source of bioactive compounds can be used as functional food supplements to deter the need of antioxidants as well as microbes related to digestive and gastrointestinal tract, which are further needed to be explored in more details. Therefore, further studies are in progress to isolate the antioxidant and antimicrobial agents from MEJR using different spectroscopic techniques.