INTRODUCTION:

Lepidium sativum L. is a herb whose seeds have been known for a high phenolic; flavonoid; and fatty acid content. It has been shown to be a good nutraceutical with favorable antioxidant; anticancer, anti-inflammatory as well as fracture-healing properties. An antioxidant is considered to offer protection against oxidative stress-related diseases, including cancer^1,2. Data from model systems has shown that reversal of inflammation (a common feature in several diseases including cancer) can be accomplished by natural molecules³. Despite the existence of in-vitro and in-vivo models, a systematic investigation of the extraction methods and correlatable antioxidants properties is lacking^4-9 No work has hitherto been reported that has systematically compared UAE; MAE and chemical (Soxhlet and crude solvent) extraction methods in terms of their bioactivity (components from Lepidium sativum L.) in certain antioxidant assays. Also, the subsequent characterization of the best extract, using UPLC and GC-MS^10,11,12 has hitherto not been done. This aspect is pertinent, since it is widely known that the activity is dependent on multiple variables, including the source of the seeds; their dormancy status; as well as the mode of extraction and the type of solvents^13,14,15. Other reports have provided some evidence for UAE and MAE-based extraction yielding bioactive components from seed sources, thereby obviating the need of using harsh, volatile, harmful solvents^{16,
17, 18}. In this regard, there have been reports wherein the methanolic as well as aqueous extract may be better than the non-polar solvents in terms of their bioactivity potential, especially for Lepidium sativum L.^{5,19,20,21,22}. For the first time, as part of our experimental flow, testing was done as an antioxidant activity-guided screening and selection approach using three well-established in vitro free radical scavenging assays (DPPH²³, FRAP and Nitric Oxide assays)^24,25. In order to identify the most plausible contributors to the observed antioxidant activity; UPLC as well as GC-MS-based analysis was done. GC-MS-based analysis has been done in the past for correlating the probable bioactive contributors to the observed bioactivity²⁶. Similarly, UPLC, another analytical tool has also been employed to identify parent compounds and their metabolites in various fluids²⁷. This study will eventually aid in the selection of optimal extraction techniques as well as the in vitro antioxidant assays for the better identification of bioactive compounds from the different extracts. Validation of our experimental flow (specifically for Lepidium sativum L.) provides us an opportunity to extend this work to further evaluate antioxidant activity as well as cytotoxicity²⁸ and cell death potential in higher order model systems (in vitro and in vivo).

MATERIALS AND METHODS:

Plant Material:

The Lepidium sativum L. seed was obtained commercially and authenticated at the National Institute of Siddha, Chromepet, Chennai, Taiml Nadu, India in consultation with an Siddha doctor Dr. Sathyarajeswaran (No: L02061802S- certificate enclosed). The seed samples were also grown in our nursery (Vellore institute of Technology, Vellore) from June to October and the plant was authenticated (ID: RVC005) by Mrs. Angelin Vijakumari, Botanist, Vorhees College, Vellore (See Supplementary information for authentication certificate). The seed was ground into a powder using a domestic mixer and grinder (Butterfly 750 watt) and stored in a sterile glass vial at -20°C.

Chemicals and Reagents:

Analytical grade n-hexane; Ethyl acetate; Methanol; HPLC grade Acetonitrile; DMSO; Folin–Ciocalteu; ascorbic acid; sodium carbonate; potassium acetate; Aluminium chloride; 2-2-Dipheny-1-picrylhydrazyl (DPPH); 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ); Ferrous sulphate; Sodium Nitroprusside (SNP); Naphthyl ethylene diamide (NED); Sulphamide; Phosphate buffer; Acetic acid from Himedia Laboratories Pvt. Ltd., India. Gallic acid, Quercetin (Sigma Aldrich, India).

Extraction of Lepidium sativum L. Seed:

Classical Soxhlet extraction (SOX):

Five (5) grams of the seed powder was taken up in 120 ml of each of the different solvents (hexane; ethyl acetate and methanol respectively) with different polarities. The protocol followed was standardized in our laboratory, with a few modifications²⁹.

Simple Crude Extract preparation (CRU):

About 0.5g (500mg) of powder was extracted with 25ml of the following solvents: hexane (CRU HEX); Ethyl acetate (CRU EA); Methanol (CRU MeOH) and Aqueous water (CRU AQU)³⁰.

Microwave-Assisted Extraction (MAE):

Five (5g) of seed powder was mixed with 120ml of aqueous distilled water. The suspension was then stirred for 20 min at room temperature in a 250ml beaker. The suspension was then irradiated for 6 minutes, using a domestic microwave (650W- LG I Wave, Model No: MH2344DB)^31,32,33.

Ultrasound Assisted Extraction (UAE):

Five (5g) of seed powder was mixed with 120mL of aqueous distilled water and the suspension was stirred for 20 min at room temperature in a 250ml beaker. Subsequently, probe sonication (20 kHz, Sonics) was done^17,34. For each of the extracts, the yield (mg/g dry weight of Seed powder) was calculated based on the formula provided below: [Dry weight of residue after extraction/Initial weight of seed powder before extraction] X100

Total Phenolic Content (TPC) and Total Flavonoid Content (TFC):

The TPC and the TFC assay was performed by using the Folin-Ciocalteu and the aluminium chloride methods respectively using well-established, published protocol. These methods have conventionally been used for the analysis of phytochemical from various plant sources^35,36.

DPPH assay:

The diphenyl-2- picrylhydrazyl (DPPH) antioxidant assay measured the free radical scavenging ability of our crude seed extracts. The antioxidant potential of our extract was compared with that of the standards (Ascorbic acid and Quercetin), based on spectrophotometric measurements at a λmax of 517nm (color change from purple to colorless)³⁷. A 0.1mM methanolic stock solution was prepared to give an absorbance of 1.238±0.2. 100µL of different concentrations (0-1000µg/mL) of Lepidium sativum L. extracts and 900µL of the 0.1M of DPPH solution were mixed and incubated for 30 minutes in the dark. % age of radical scavenging activity = (Abs control - Abs sample)/Abs control × 100.

FRAP assay:

The FRAP assay is a simple and sensitive experiment to measure the reduction of Fe3+ µM to Fe2+ µM in Ferric3+- tripyridyl-triazine mixture to blue color ferrous complex. A freshly prepared FRAP solution contained a mixture of 300mM acetate buffer (pH 3.6); 10mM TPTZ (in 40mM HCl) and 20mM ferric chloride solution (FeCl3·6H2O) in 10:1:1 (v/v) ratio and this solution was pre-warmed to 37°C. The reducing ability was measured by dissolving 50µL of different concentrations of standard Lepidium sativum L. seed extracts (0-1000µg/mL) with 50µL water. Then, 900µL of FRAP reagent was added and the solutions were incubated in the dark (4 min). This change in absorbance was measured at 593nm and the FRAP value determined based on the standard curve generated using different concentrations of aqueous ferrous sulphate (0-1000µM -FeSO4.7H2O). The FRAP potential was expressed as the micromolar concentrations of Fe3+ that were reduced to Fe2+ (μM/L) and the results compared with that of ascorbic acid and quercetin standards³⁷.

Nitric Oxide assay:

The NO scavenging capacity of Lepidium sativum L. seed extracts was observed by the light-mediated conversion of Sodium Nitroprusside (SNP)-mediated nitric oxide to nitrite ion. These nitrite ions were estimated using the Griess reagent at a λ_max of 542 nm^38,
39. Briefly, the Greiss reagent solution (C) was prepared by mixing two different solutions A and B at a ratio of 1:1 v/v. Solution A contained 2% (% w/v) sulfanilamide and 4% (% w/v) H₃PO₄. Solution B contained 0.2% (% w/v) NED. The standard curve was obtained using different concentrations of sodium nitrite (0-30µM/mL) in aqueous solution. The reaction was initiated by preparing 0.5ml of 10mM freshly prepared SNP in 20mM Phosphate buffer, pH 7.4. In this solution, different concentrations (0-1000µg/ml) of seed extracts were incubated at room temperature under light for 150 min. Water without samples were used as the controls with quercetin as the standard. One (1) ml of Greiss reagent (Solution C) was added to each sample to form a pink azo dye and the absorbance was read at 542nm. The % NO scavenging was calculated by [(NO₂- in control - NO₂-samples)/ NO₂- in control] x 100.

Statistical analysis:

All the experiments were carried out in triplicate. Mean; standard deviation, Spearman Rank Correlation was analysed in EXCEL 2016; Graph Pad prism 8.0, trial version.

GC MS analysis of methanol extract of Lepidium sativum L.:

GC-MS analysis of the methanolic Lepidium sativum L. seed powder was outsourced to Hubert Enviro Care Systems Private Limited, Chennai, Tamil Nadu.

UPLC-UV-PDA analysis of methanol extract of Lepidium sativum L.:

One hundred (100) mg of seed powder was extracted using four (4) ml of 90% methanol containing 0.5% Acetic acid was prepared and vortexed for 15min. Ultrasonication step was done for 1 minute and repeated three times (100% amplitude with 2 sec on 1sec off). The supernatant was centrifuged for 10 minutes at 6000 rpm and the samples were dried in a rotary evaporator. The crude extract was dissolved with 1ml of DMSO and filtered through 0.22µm syringe filter before their UPLC analysis. The Ultra pressure liquid chromatography H class Acquity, waters LC system was coupled with a Photo diode array detector (PDA) spectrum range (210-700nm), Acquity UPLC H-ss T3 2.1X100mm; 1.8µm column; temperature at 45°C. A 2µl sample was injected for a total run time of 16 minutes with a flow rate at 0.5ml/min. The mobile phases A and B were respectively Water +0.1% acetic acid as well as Acetonitrile + 0.1% acetic acid. gradient elution profile followed: for 0-1 min- 1% B; 10 min- 30% B; 12 min-95% B; 12.1 min -16th min-1% B. Prior to each run, the chromatographic system was equilibrated with 1% B for 30 min. The retention times (RT) and spectra (λ_max) was recorded by using Empower® 3 software and this enabled us to correlate the presence of phenolic and flavonoid compounds⁴⁰.

RESULTS:

Extraction, TPC and TFC:

Extraction was done based on our defined conditions and a qualitative and quantitative phytochemical analysis was performed for the total phenolic and flavonoid content. The yield, nature and color of the solvent-free extract as well as the total phenolic and flavonoid content are shown in (Table 1). Barring a few exceptions, the exaction yield was not significantly different between the extraction technique and solvent type. The total CRU-MeOH extract had high phenolic content of 4464.1±349.7mg GAE/100g while that of SOX-MeOH was 3824.4±90mg GAE/100g. The green extraction method of CRU-AQU, MAE-AQU and UAE-AQU also showed the presence of phenolic content 2878.8±245.0; 2601.0±111.4; 2401±166.3mg GAE/100g respectively. In terms of the TFC, UAE-AQU was found to have a high value of 1520.6±182.2mg QUE/100g followed by MAE-AQU value of 1332.5±62mg QUE/100g. The SOX-MeOH; CRU-MeOH and CRU-AQU was found to be 927.8±72.9; 773.2±291.6 and 541.2±109.3mg QUE/100g respectively.

Table 3: Compounds present in the methanolic extract of L. sativum seeds, ^aCompounds detected in total ion chromatogram Fig. 4, ^b new compounds detected in the methanolic extract of the L. sativum seed

Sl. No.	RT	Name	Mass	Area %
1	6.57^a	Benzyl nitrile	117.1	17.19
2	11.3^a	Benzene, (isothiocyanatomethyl)-	149	4.65
3	15.41^a	3',5'-Dimethoxyacetophenone	180.1	6.46
4	21.9^a	Hexadecanoic acid, methyl ester	270.3	2.35
5	22.54	n-Hexadecanoic acid	256.2	1.83
6	22.98	Hexadecanoic acid, ethyl ester	284.3	1.74
7	24.42	9,12-Octadecadienoic acid (Z,Z)-, methyl ester	294.3	2.98
8	24.5	9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-	292.2	9.61
9	24.52	6-Octadecenoic acid, methyl ester, (Z)-	296.3	6.12
10	24.98	Methyl stearate	298.3	0.46
11	25.23^a	cis-Vaccenic acid	282.3	22
12	25.42	9,12-Octadecadienoic acid, ethyl ester	308.3	2.47
13	25.51	9,12,15-Octadecatrienoic acid, ethyl ester, (Z,Z,Z)-	306.3	11.66
14	25.96	Octadecanoic acid, ethyl ester	312.3	0.43
15	27.4^a	cis-11-Eicosenoic acid, methyl ester	324.3	2.02
16	27.81^b	Methyl 18-methylnonadecanoate	326.3	0.37
17	28.35^b	2-Butenoic acid, 2-methyl-, 2-(acetyloxy)-1,1a,2,3,4,6,7,10,11,11a-decahydro-7,10-dihydroxy-1,1,3,6,9pentamethyl-4a,7a-epoxy-5Hcyclopenta[a]cyclopropa[f]cycloundecen-11-yl ester, [1aR-[1aR,2R,3S,4aR,6S,7S,7aS,8E,10R,11R(E),11aS]]-	490.3	1.06
18	29.91	[2-(Benzo[4,5]thiazolo[2,3-c][1,2,4]triazol-3ylsulfanyl)ethyl](dimethyl)amine	278.1	2.01
19	31.03^b	5H-Cyclopropa[3,4]benz[1,2-e]azulen-5-one,3,9,9atris(acetyloxy)-3-[(acetyloxy)methyl]-2-chloro-1,1a,1b,2,3,4,4a,7a,7b,8,9,9a-dodecahydro-4a,7b-dihydroxy-1,1,6,8-tetramethyl-, [1aR-(1a.alpha.,1b.beta.,2.alpha.,3.beta.,4a.beta.,7a.alpha.,7b.alpha.,8.alpha.,9.beta.,9a.alpha.)]-	584.2	0.27
20	32.14^{a b}	7,8-Epoxylanostan-11-ol, 3-acetoxy-	502.4	0.7
21	32.77^b	17-(1,5-Dimethylhexyl)-10,13-dimethyl-3styrylhexadecahydrocyclopenta[a]phenanthren-2-one	488.4	0.31
22	36.06^b	1H-Cyclopropa[3,4]benz[1,2-e]azulene-2,5-dione, 9,9abis(acetyloxy)-3-[(acetyloxy)methyl]-1a,1b,4a,7a,7b,8,9,9aoctahydro-4a,7b-dihydroxy-1,1,6,8-tetramethyl-, [1aR-(1a.alpha.,1b.beta.,4a.beta.,7a.alpha.,7b.alpha.,8.alpha.,9.beta., 9a.alpha.)]-	504.2	0.28
23	36.66	gamma.-Sitosterol	414.4	1.06
24	36.76^{a b}	Ergosta-14,22-dien-3-ol, acetate, (3.beta.,5.alpha.)-	440.4	0.22

UPLC-UV-PDA analysis of methanol extract of Lepidium sativum L.

The methanolic extracts of Lepidium sativum L. were analysed by UPLC-PDA. The standard quercetin at 302.24µg/mL shows RT-8.531±0.02 and a maximum absorbance at 369.9 nm shown in (Fig. 5(A)). The 7 peaks detected in the crude methanolic extract of Lepidium sativum L. seeds had a RT of 5.9, 6.804, 6.238, 6.436, 6.69, 7.07, 8.55 were identified and correlated with that of standard quercetin (Fig.5 (B)) and (Table 4). The spectral analysis using UV-VIS PDA detector shows the maximum absorbance of 351.9nm, 328.5nm, 330.9nm, 333.3nm, 328.5nm, 368.7nm respectively confirmed the presence of phenolic and flavonoid compounds.

Table 4: Analysis of UPLC data -Correlation of area of methanol crude extract with standard area under curve (AUC) Quercetin equivalents and possible compounds. GAE- Gallic acid, QUE- Quercetin, AUC- Area under Curve, ND- Not determined

Sl. No.	Name of compound	RT	λ max (nm)	Area	Correlation AUC R² = 0.9959/y = 13188x (µg QUE/ 100mg) seed
1	p-Coumaric acid	5.9	351.9	1804	0.1
2	ND	6.084	328.5	33105	2.5
3	ND	6.238	330.9	1453	0.1
4	ND	6.436	333.3	6040	0.5
5	Ferulic acid	6.69	327.3	8297	0.6
6	ND	7.07	328.5	1447	0.1
7	Quercetin	8.555	368.7	9869	0.7

DISCUSSION:

Lepidium sativum L. is known for its high polyphenolic and flavonoid content (Table 1). CRU MeOH had a relatively higher TPC of 4464.1±349.7 mg GAE/100g followed by SOX MeOH which had a content of 3824.4±90.0 mg GAE/100g. Despite variations in the TPC content and protocol-related differences in the experimental conditions, similar results were reported by others²⁰. Such variations in TPC may be attributable to differences in the solvent, time, temperature and seed texture (dehulled/milled whole seed). TPC content following microwave processing was 2601.0±111.4 and was at variance with published data^12,41. Hence, there is a need to validate protocols across laboratories within the country and globally. Non polar extracts also exhibited phenolic content. This finding is similar to the results reported by others⁴².We have demonstrated qualitatively similar findings in terms of the TFC values (Table 1). Again, there are variations in the TFC content reported by others ^{6, 43, 44}. The TFC content in the CRU MeOH was 2457 ± 281 mg QUE/100 g and proves the capabilities of this solvent to extract flavonoids. TFC measurements could not be obtained for the non-polar extracts due to turbidity. Quantitative variations in TPC and TFC content may be attributable to differences in the methodology; geographic source of the seeds and dormancy status^{45, 46}.

The different extracts were evaluated using DPPH, FRAP and NO-based assays (Fig.1), (Fig. 2), (Fig. 3) in order to identify the best extract in terms of their optimal free radical scavenging potential. This evaluation was based on comparisons with the data reported by others and is discussed below. DPPH scavenging potential of a 100µg/ml soxhleted Lepidium sativum L. methanol seed powder extract was 11.63±0.3¹⁹. The 53% Lepidium sativum L. ethanol seed extract, showed an IC50 value of 162.4±2.3µg/mL⁶. The fractioned n-butanolic extract of Lepidium sativum L. exhibited an IC50 of 67.1±0.3µg/mL⁴⁷. The soxhleted Lepidium sativum L. methanolic seed extract showed an IC50 value of 85µg/ml (similar to our IC50 value of 50.61µg/mL). Also, this paper has shown that the soxhleted non-polar n-hexane extract has an IC50 value of 1415µg/ml, while that of the ethyl acetate fraction was 215µg/mL⁴⁴. Data is currently unavailable on the DPPH activity of aqueous extracts of Lepidium sativium L. seed powder. However, UAE data on the Beinncas hipida seeds extracted in the presence of 99.5% methanol showed an inhibition of DPPH assay-based free radical production by 32.12±0.38%, while the conventional Soxhlet-based extraction showed a scavenging potential of 28.70±0.7%⁴⁸. This finding substantiates our experimental design in terms of incorporating the UAE method as a possible eco-friendly strategy. Our DPPH-based IC50 value determination also matched qualitatively with that reported for scavenging by the methanolic extract of the pomegranate seed⁴⁹. Our positive results provided a sound basis for evaluating the possible role of our extract in quenching other free radicals.

The ability of electron transfer for Lepidium sativum L. extracts was performed by FRAP assay shown in (Fig. 2). This crude methanolic extract exhibited the highest free radical scavenging based on the FRAP assay. At 100µg/mL, it showed a FRAP value of 217.82±12.82. However, in another study involving the soxhleted 80% methanolic Lepidium sativum L. extract, a FRAP value of 1317.04 ± 5.74µmol Fe++/g¹² was reported. The n-butanolic fraction of Lepidium sativum L. showed a maximum reducing power of 100±0.051µg/mL⁴⁷. Callus cultures of Lepidium sativum L. grown under white light produce has the maximum scavenging activity equivalent to 629.78µM (Trolox C equivalent)⁴³. For better visualization and correlation with our reported DPPH assay-based IC50 value determinations, we have reported the relative antioxidant potential of our extracts, based on our interpolated values. The values respectively for the CRU MeOH; SOX MeOH; CRU AQU; MAE AQU; UAE AQU were 114.13; 77.13; 46.20; 56.83; 53.76 FRAP value. There are other reports, wherein MAE and UAE-based extraction of ethanol/water 50:50v/v of Coriandrum sativum has shown 68765 and 1198 mmol Trolox®/100g⁵⁰. This shows that Lepidium sativum L. extracts exhibit good reducing power potential. However, variations in the absolute values perforce warrant inter-laboratory validation of standardized protocols.

Ability of bioactive compounds to scavenge nitric oxide in biological system was also evaluated. Nitric oxide scavenging potential of Lepidium sativum L. is shown in (Fig. 3). The NO scavenging potential of CRU MeOH found to be 296.74µg/mL. Similar results were obtained from the soxhleted methanol extract of Lepidium sativium L. with a value of 132µg/mL⁴⁴. There are reports of novel compounds (kaempferol-3-O-(2-O-sinapoyl)- β-D-galactopyranosyl-(1→2)-β-D-glucopyranoside-7-O-α-L-rhamnopyranoside and quercetin-3-O-(6-O-benzoyl)-β-D-glucopyranosyl-(1→3)-β-D-galactopyranoside-7-O-α-L- rhamnopyranoside extracted from Lepidium sativum L. seeds. Under their experimental conditions in a cell-based study, these compounds respectively showed an IC50 value of 25.36 µM and 25.08 µM in terms of their nitric oxide scavenging potential. This NO was generated following exposure of RAW264.7 macrophage cell line to Lipopolysaccharide⁵¹. Again, a comparison based on interpolation was done at the DPPH assay-based IC50 value for comparing the NO scavenging potential with that of the results obtained from the DPPH and FRAP assays. CRU MeOH shows (8.53%), SOX MeOH-(6.33%), CRU AQU (5.97%), MAE AQU (5.85%), UAE AQU (53.76%). As observed in the TFC method, the NO assay could not be performed for the non-polar extracts (turbidity-related issues). This turbidity precluded us from obtaining meaningful, spectrophotometric-based results.

Our study showed that all extracts were positively correlated (r) between TFC and TPC and the antioxidant assays (Table 2). Similar correlations has been reported (by others) in the case of certain crude seed extracts for TPC and Antioxidant capacity (DPPH, FRAP and TEAC), while other plant sources did not exhibit this type of a positive statistical trend⁵². There are reports, wherein there is a strong correlation between Total phenolic content and DPPH, when compared to non-tannic phenolics. These assays were performed with bioactive components extracted using 70% acetone from each of the twenty four (24) different cultivated and wild plant species⁵³. Another report on the ethanolic extracts of freeze-dried and air-dried samples of Chenopodium quinoa showed a very good correlation between TPC and TFC and DPPH and the FRAP assays⁵⁴. This shows that the correlation between TPC, TFC and antioxidant capacity may be specific to the different stages of plant growth, as well as experimental variations in the respective extraction protocols. Such variations reported by others, again underscore the imperative need to validate our experiments using globally accepted methods.

Our design also involved GC-MS (Fig. 4; Table 3) and UPLC-based (Figure 5A and 5B; Table 4) analytical characterization of the probable bioactive components (singly and/or in combination) that may have contributed to the hitherto unreported correlation with the antioxidant activity discussed below. Our experimental design as well as the methodologies has been validated since, similar compounds have been reported in previous studies, albeit in a qualitative manner. Barring 23 minor compounds, 7,10-Hexadecadienoic acid, methyl ester has been detected in the Lepidium sativum L. seed oil¹¹. Further, along with 11 minor compounds, 9,12,15-octadecatrienoic acid (Alpha-linolenic acid) has been reported in the Lepidium sativum L. seed extracts¹⁰. As has been detected in our study, Cis-vaccenic acid has been reported in the methanolic extract of Lepidium sativum L., even though the analysis was done on the leaves of this plant⁵⁵. Apart from the crude methanolic extracts, microwave-assisted extraction of Carum carvi L. seed` has shown the presence of 14 compounds in comparison with the detection of 6 compounds in the case of the classical solid-liquid extraction⁵⁶. In this context, it is pertinent to also point out that there are a few compounds isolated from Lepidium sativum L. seeds which have hitherto not been reported (Table 4).

To validate the presence of bioactive compounds and correlation with TPC, TFC and antioxidant assays, Lepidium sativum L. methanolic extracts were analysed using Acquity UPLC H-Class coupled with a PDA detector. This type of an analytical tool is considered to be better than the conventional HPLC in terms of the sensitivity and the time taken for the analysis to be completed⁵⁷. The retention time of our Gallic Acid reference molecule (Rt-1.40±0.01) matched that reported in the literature⁵⁸. Also, quercetin demonstrated a remarkable correlation of the retention time (Rt- 8.53±0.019) with that found in our crude methanolic extract (Fig. 5(A). There are 7 major peaks found in Lepidium sativum L. extract as shown in Fig. 5(B). Again, a striking correlation was seen with the retention time data reported in the literature for p-coumaric acid (Rt-5.9), ferulic acid (Rt-6.69)⁵⁸. Concomitant UV-visible spectroscopy-based measurements using our UPLC system provided another line of evidence to further substantiate the presence of p-coumaric acid; ferulic acid and quercetin with the absorbances (verified) respectively being 351.9 nm, 327.3 nm and 368.7nm. The other RT peaks (6.084, 6.249, 6.436, 6.549, 7.070) showed maximum absorbance at 328.5 nm, 330.9 nm, 333.3 nm, 308.2 nm, 328.5nm respectively. Since the maximal absorbance of these 5 peaks (UPLC retention time data) matched that reported for cinnamic acid esters as well as the flavonol glycosides, it can be inferred that our crude methanolic extract may also contain these compounds^{30, 59, 60}. LC-MS- based analyses showed that there was a qualitative correlation in terms of the presence of quercetin, p-coumaric acid and ferulic acid^6,7,43. Our positive findings substantiate the design adopted by us in this study.

CONCLUSIONS:

Physical methods like MAE and UAE have shown potential for the extraction of important bioactive principles from the Lepidium sativum L. seeds. Also, crude methanolic and crude aqueous extracts have good antioxidant and radical scavenging potential. Hence, our findings substantiate the “green-chemistry” approach for the isolation and development of molecules with antioxidant potential. Our positive results pave the way for optimizing our experimental conditions to validate our antioxidant data as well as possibly extend our studies to determining bioactivity of our Lepidium sativum L. seed extracts in higher order model systems.

LIST OF ABBREVIATIONS:

DMSO-Dimethyl sulfoxide, SOX HEX-Soxhlet Hexane extract, SOX EA-Soxhlet Ethyl acetate extract, SOX MeOH- Soxhlet methanol extract, CRU HEX- Simple crude extract-Hexane, CRU EA- Simple crude extract Ethyl acetae, CRU MeOH-Simple crude extract Methanol, CRU AQU-Simple crude extract-distilled water, UAE AQU-Ultrasound assisted extraction water, MAE AQU-Microwave assisted extraction water, GAE- Gallic acid Equivalent, QUE-Quercetin Equivalent, DPPH-2,2-diphenyl-1-picrylhydrazyl, FRAP-Ferric reducing ability of plasma, NO-Nitric oxide, UPLC- Ultra performance Liquid chromatography, AUC-Area under curve, GC-MS-Gas chromatography-Mass spectrometry.

AUTHOR’S CONTRIBUTION:

The primary author was involved in the bench work in terms of standardizing experimental conditions and generation of data. Also, he was involved in writing the first draft of the manuscript. The corresponding author was involved in project conceptualization; drafting the experimental design and trouble-shooting. Also, he was involved in editing the manuscript and provides critical inputs and insights for better analysis and interpretation of the data.

ACKNOWLEDGEMENT:

The authors acknowledge the infrastructural support and constant encouragement provided by the management of VIT, Vellore. Also, the authors thank Dr. Sathiya Rajeswaran, Siddha Research Scientist, Siddha Central Research Institute, Chennai for providing critical insights in terms of the clinical significance of Lepidium sativum Linn. and for authenticating the seeds. We also thank DST (SERB) for funding a project on drug delivery systems ((SB/SO/HS-157 (2013)) and for creating the scientific ambience, that served to encourage all the research scholars and the project students in the laboratory. We thank Hubert Enviro Care Systems Private Limited, Chennai, Tamil Nadu for the GC-MS analysis outsourced to them.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

FUNDING:

This work was supported, in part, by the Vellore Institute of Technology. Also, Council of Scientific and Industrial Research (CSIR), New Delhi, has provided additional financial support in the form of a Senior Research Fellowship (09/844(0075)/2019-EMR 1) to the lead author.

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Received on 20.04.2020 Modified on 29.06.2020

Research J. Pharm. and Tech. 2021; 14(6):3082-3092.

DOI: 10.52711/0974-360X.2021.00539

Sl. No	Extraction Type	Total Yield (%)	SD	Nature of Extract	Colour of Extract	Total Phenolic content (mg GAE/100g) Mean ± SD	Total Flavonoid content (mg QUE/100g) Mean ± SD
1	SOX HEX	23.33	4.41	Oil	Dark yellow	1111.1±226.3	ND
2	SOX EA	25.60	1.51	Oil	Dark yellow	2081.9±243.0	ND
3	SOX MeOH	19.87	1.36	Crude paste	Burnt caramel	3824.4±90.0	927.8 ±72.9
4	CRU HEX	33.33	5.77	Oil	Dark yellow	1484.3±670.5	ND
5	CRU EA	33.33	5.77	Oil	Dark yellow	1989.3±295.5	ND
6	CRU MeOH	23.00	1.73	Crude paste	Burnt Caramel	4464.1±349.7	773.2 ±291.6
7	CRU AQU	26.67	5.77	Dried powder	Pale yellow	2878.8±245.0	541.2 ±109.3
8	UAE AQU	21.80	1.93	Dried powder	Pale yellow	2401.8±166.3	1520.6 ±182.2
9	MAE AQU	25.13	1.17	Dried powder	Pale yellow	2601.0±111.4	1332.5 ±62.0


*Fig. 1: DPPH radical scavenging assay of Lepidium sativum* Linn. Extracts**	*Fig. 2: FRAP reducing Potential of Lepidium sativum* Linn. extracts**

*Fig. 3: Nitric oxide scavenging potential of Lepidium sativum* Linn. extracts**	*Fig. 4: Total Ion Chromatograph of GC-MS analysis from methanolic extract of Lepidium sativum* Linn.**

	Spearman r	Extracts equivalent to TPC GAE (µg/ml) with Antioxidant assays			Equivalent to TFC QUE (µg/ml) with Antioxidant assays
Sl. No.	*L. sativum* extracts	DPPH	FRAP	NO	DPPH	FRAP	NO
1	SOX-MeOH	1***	1***	0.9019***	0.9129***	0.9374***	0.8574**
2	CRU-MeOH	1***	0.9879***	0.9265***	0.9129***	0.9374***	0.8574**
3	CRU-Aqu	1***	1***	0.9878***	0.9129***	0.9374***	0.949***
4	MAE	1***	1***	0.9878***	0.9129***	0.9374***	0.949***
5	UAE	1***	0.9879***	0.9878***	0.9129***	0.9374***	0.949***


Fig. 5 (A): UPLC-PDA chromatograph of standard Quercetin (302.24 µg/mL), RT-8.555, runtime-16 min	Fig. 5 (B): UPLC –PDA chromatograph of methanol extract of Lepidium sativum Linn. For RT, λmax, Area, AUC data see Table.4.