ABSTRACT
For the extraction and isolation of cis-n-docos-15-enoic acid (1), n-dodecanoyl-O-ß-D-glucuranopyranoside (2), n-tetradecanoyl-O-ß-D–arabinopyranoside (3) and n-eicosanoyl-O-ß-D–arabinopyranoside (4) from the seeds of L. culinaris.
UV spectra were measured with a Lambda Bio 20 Spectrophotometer in methanol. The 1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded on a Bruker ARX-Spectrometer, using CDCl3 and DMSO-d6 as solvents and TMS as an internal standard. Melting points were determined by a thermoelectrically heated Perfit melting point apparatus without correction. Infra Red (IR) spectra were recorded using KBr pellets with a Jasco FT-IR-5000 Spectrometer. Mass-spectrometric detection was carried out on ESI MS (Q-TOF-ESI), an electrospray-ionisation (ESI) technique with positive ionization mode. Column chromatography was performed on silica gel, 60-120 mesh. Pre-coated Aluminum TLC plates of silica gel 60 F254 were used to run and spots were visualized by exposure to iodine vapors and UV radiations and spraying with anisaldehyde- sulfuric acid solution.
Compound 1, a fatty acid, was obtained as a semisolid mass from chloroform as eluants. It decolorized bromine water solution and produced effervescence with sodium bicarbonate solution indicating unsaturated nature of a the fatty acid.Compound 2, designated as lauryl glucuranoside, was obtained as a yellowish sticky mass from chloroform-methanol (9:1) as eluants. Compound 3, myristyl-O-ß-D–arabinoside, was obtained as a colorless sticky mass from chloroform-methanol (17:3, v/v) eluants. Compound 4, an arachidyl arabinoside, was obtained as a brownish sticky mass from chloroform- methanol (9:1) eluants.
These compounds may be responsible for the quality control purposes and may be use to identifying the authentic samples from adulterated samples.
Keywords: Lens culinaris, seeds, methanolic extract, Isolation.

INTRODUCTION

Lens culinaris Medik (Leguminosae), is an ancient crop of classical Mediterranean civilization and continually plays a vital role in the global human health. It is commonly known as lentil, (Zohary and Hopf, 2000), Adas, Masur, Mercimek and Heramame (Summerfield and Muehlbauer, 1982). Lens-shaped seeds are smooth, about 4 mm in thickness, grayish-brown and faintly mottled, cotyledons pink; testae are characteristic. Its powder is cream colored; shows black particles due to pieces of testa; fragments of thick-walled, elongated, oval to polygonal cells. Its action is as constipation, flatulent, antidiuretic, and antiemmenagogue (Anonymous, 2009). It is mainly cultivated for its edible seeds (Summerfield and Muehlbauer, 1982). As stated by Hippocrates’ (460-370 BC), “Let your food be your medicine and let your medicine be your food” (Diplock et al., 1999), its seeds are an excellent source of proteins, fiber, vitamins, minerals (Chibbar et al., 2010), carbohydrate and fat (Adsule et al., 1989).
It possesses high content of saponins in the plant and has considerable interest which shows diverse biological properties (Fenwick et al., 1991). It causes toxicity to fishes (Dorsaz et al., 1988) and inhibits the growth and sporulation of a wide range of fungi (Gestetner et al., 197; Wang et al., 2007). They potentiated lysis of erythrocytes (Khalil and El-Adawy, 1994), reduced plasma cholesterol levels (Sidhu and Oakenfull, 1986), showed anticancer activity (Konoshima et al., 1992), an inhibitory effect on the human immunodeficiency virus (Nakashima et al., 1989) and antioxidant potential (Amarowicz et al., 2001). Previous phytochemical investigations revealed the presence of steroidal glycosides (Price et al., 1987), saponins (Fenwick et al., 1991), hydroxyarginine (Bell et al., 1999), phenolic compounds (Amarowicz et al., 2001), 4-chloro-1H-indole-3-n-methylacetamide, itaconic acid, arbutin, gentisic acid 5-O-glycoside, and phytosterols (Benveniste, 1986; Tsopmo and Muir, 2010).
A new aromatic ester 3′-methyl–n-pentadecal benzoate along with ß-sitosteryl-n-octadec-9′-enoate, n-tetradecanyl linoleiate and n-octatriacosanoic acid has been isolated from a methanolic extract of the seeds of plant (Jameel et al., 2014). Some new antioxidant compounds like ß-sitosteryl-3-(2′-n-eicosanyloxy)-benzoate, n-octadec-9-enoyl-1-ß-D-glucurano-pyranoside, a-D-galactopyranosyl-(6?1′)-a-D-galactopyranosyl-(6′?1”)-a-D-galactopyranosyl-(6”?1”’)-a-D-galactopyranoside, and benzoyl-O-a-D-glucopyranosyl-(2a?1b)-O-a-D-glucopyranosyl-(2b?1c)-O-a-D-glucopyranosyl-(6c?1d)-O-a-D- glucopyranosyl- (6d?1e)- O-a-D- glucopyranoside, along with n-heptadecanyl n-octadec-9-anoate and ß-sitosterol have also been isolated from the methanolic extracts of the seeds of the seeds (Jameel et al., 2015). Due to valuable contribution to human health globally, isolation of new phytoconstituents has been targeted in this communication from the seeds of L. culinaris grown in Delhi region.

MATERIALS AND METHODS

UV spectra were measured with a Lambda Bio 20 Spectrophotometer (Perkin Elmer, Rotkreuz, Switzerland) in methanol. The 1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded on a Bruker ARX-Spectrometer (Rheinstetten, Baden-Wurttemberg, Germany), using CDCl3 and DMSO-d6 as solvents and TMS (Fluka analytical, Sigma-Aldrich, Zwijndrecht, Netherland) as an internal standard. Melting points were determined by a thermoelectrically heated Perfit melting point apparatus (Ambala, India) without correction. Infra Red (IR) spectra were recorded using KBr pellets with a Jasco FT-IR-5000 Spectrometer (FTS 135, Kawloon, Hong Kong). Mass-spectrometric detection was carried out on ESI MS (Q-TOF-ESI) (Waters Corp., Manchester, UK), an electrospray-ionisation (ESI) technique with positive ionization mode. Column chromatography was performed on silica gel (Qualigens, Mumbai, India), 60-120 mesh and solvents taken were purchased from Merck Specialties (E. Merck, Pvt. Ltd. New Delhi, India). Pre-coated Aluminum TLC plates of silica gel 60 F254 (Merck, Darmstadt, Germany) were used to run and spots were visualized by exposure to iodine vapors and UV radiations and spraying with anisaldehyde- sulfuric acid solution.
Plant material
The L. culinaris seeds was collected from the Herbal Garden of Jamia Hamdard, New Delhi and identified by Prof. M.P. Sharma, incharge Herbal garden. A specimen voucher of the drug was deposited to the Phytochemistry Research Laboratory, Jamia Hamdard with a reference number PRL-JH/2011/05.
Preparation of crude extract and isolation
The dried L. culinaris seeds (3.5 kg) were coarsely powdered and extracted with methanol for 72 hr using a Soxhlet extractor. The extract was dried under reduced pressure to obtain a dark brown residue (230 g, yield 0.0657%). The residue (100 g) was dissolved in minimum amount of methanol and adsorbed on column grade silica gel (60-120 mesh) to obtain slurry. The slurry was dried in air and chromatographed over silica gel column loaded in petroleum ether. The column was eluted with chloroform and chloroform-methanol (9:1 and 17:3, v/v) mixture to isolate the compounds 1-4.

RESULTS

cis-Docos-15-enoic acid (1)
Elution of the column with chloroform gave a semisolid mass of 1, 31g (0.885 % yield), UV ?max (MeOH): 205 nm (log 2.3), IR ?max (KBr): 3347, 2884, 1702, 1625, 1435, 1379, 1219, 1052, 721 cm-1. 1HNMR (CDCl3); d 5.34 (2H, m, w1/2=8.8 Hz, H-15, H-16), 2.76 (2H, m, H2-2), 2.27 (2H, m, H2-14), 2.03 (2H, m, H2-17), 1.57 (2H, m, CH2), 1.29 (8H, brs, 4 x CH2), 1.25 (20H, brs,10 x CH2), 0.87 (3H, t, J=6.5 Hz, Me-22). ESI-MS m/z (ret.int): 338 [M]+ (C22H42O2) (10.2), 111 (38.1).
Lauryl-O-ß-D-glucuranoside (2)
Elution of the column with chloroform-methanol (9:1, v/v) afforded a yellow semisolid mass of 2, purified by preparative TLC (chloroform-methanol, 17:3, v/v), 3 g (0.085 % yield); UV ?max (MeOH): 207, 259 mm (log 2.3, 1.2). IR ? max (KBr): 3495, 3387, 3261, 2897, 1721, 1707, 1624, 1433, 1322, 1179, 1122, 1080 cm-1; 1HNMR (CDCl3): d 5.26 (1H, d, J=7.5 Hz, H-1′), 4.64 (1H, m, H-5′), 4.06 (1H, m, H-2′), 3.77 (1H, m, H-3′), 3.65 (1H, m, H-4′), 2.29 (2H, m, H2-2), 2.02 (2H, m, CH2), 1.58 (2H, m, CH2), 1.25 (14H brs, 7 x CH2), 0.87 (3H, t, J=6.3 Hz, Me-12). ESI MS m/z (ret.int); 376 [M]+ (C18H32O8) (15.2), 193 (8.7), 183 (6.9).
Myristyl-O-ß-D–arabinoside (3)
Elution of the column with chloroform-methanol (17:3, v/v) yielded a colorless semisolid mass of 3, crystallized from chloroform-methanol (1:1), 10 g, (0.285 % yield), Rf UV ?max (MeOH): 207 nm (log 2.8); IR ?max (KBr): 3450, 3353, 3266, 2896, 1735, 1620, 1432, 1321, 1182, 1060, 725 cm-1. 1HNMR (CDCl3): d 5.23 (1H, d, J=7.5 Hz, H-1′), 3.85 (1H, m, H-2′), 3.67 (1H, m, H-3′), 3.44 (1H, m, H-4′), 3.36 (2H, d, J=6.8 Hz, H2-5′), 2.69 (2H, t, J=7.9 Hz, H2-2), 2.25 (2H, m, CH2), 1.53 (2H, m, CH2), 1.25 (18H brs, 9 x CH2), 0.88 (3H, t, J=6.5 Hz, Me -14); ESI MS m/z (ret.int): 360 [M]+ (C19H36O6) (15.11), 183 (7.1), 149 (25.6), 133 (10.3).
Arachidyl-O-ß-D-arabinoside (4)
Elution of the column with chloroform-methanol (9:1, v/v) furnished a brown semisolid mass of 4, purified by preparative TLC (chloroform-methanol, 17:3, v/v), 5g (0.142 % yield), UV ? max (MeOH): 210 nm (log 2.3); IR ? max (KBr): 3386, 3225, 2909, 2841, 1724, 1644, 1449, 1365, 1229, 1061, 804, 716 cm-1. 1H NMR (CDCl3); d 5.28 (1H, d, J = 7.8 Hz, H-1′), 4.29 (1H, m, H-2′), 4.06 (1H, m, H-3′), 3.70 (1H, m, H-4′), 3.34 (2H, d, J=6.9 Hz, H2-5′), 2.69 (2H, t, J=7.5 Hz, H2-2), 2.23 (2H, m, CH2), 1.96 (2H, m, CH2), 1.51(2H, m, CH2), 1.21 (8H brs, 4 x CH2), 1.18 (20H, brs,10 x CH2) , 0.81(3H, t, J=6.5 Hz, Me-20). ESI -MS m/z (ret.int); 444 [M]+ (C25H48O6) (2.3).

Figure 1. Compounds 1-4 isolated from methanolic extract of seeds of Lens culinaris Medik. Click here to view full image

DISCUSSION

Compound 1, a fatty acid, was obtained as a semisolid mass from chloroform as eluants. It decolorized bromine water solution and produced effervescence with sodium bicarbonate solution indicating unsaturated nature of a the fatty acid. It showed IR absorption bands for the carboxyl group (3347 cm-1, 1702 cm-1) and a long chain aliphatic hydrocarbon (721 cm-1). Its Mass spectrum showed a molecular ion peak at m/z 338 corresponding to a molecular formula of a fatty acid, C22H42O2. The daughter ion peak arising at m/z 111 [M-227, CH3(CH2)5CH=CH+] indicated the presence of unsaturation at C-15. The 1H NMR spectrum of 1 exhibited a two-proton multiplets at d 5.34 (w1/2 8.8 Hz) assigned to cis-oriented vinylic H-15 and H-16 protons, respectively. Three two-protons multiplets at d 2.76, 2.27 and d 2.03 were ascribed to methylene H2-2 protons adjacent to the carboxylic group and H214 and H2-17 protons nearby to the vinylic carbons. The remaining methylene protons resonated between d 1.57-1.25. A three-proton triplet at d 0.87 (J=6.5 Hz) was attributed to primary C-22 methyl protons. On the basis of above discussion compound 1 is characterized as cis-docos-15-enoic acid. This is new long chain aliphatic acid.
Compound 2, designated as lauryl glucuranoside, was obtained as a yellowish sticky mass from chloroform-methanol (9:1) as eluants. It gave positive tests for glycosides and displayed characteristic IR absorption bands for hydroxyl groups (3495, and 3387 cm-1), carboxylic function (3261 and 1707 cm-1), and ester function (1721 cm-1). On the basis of mass spectral data its molecular ion peak was determined at m/z 376 corresponding to the molecular formula of an acyl glycoside, C18H32O8. The ion peaks arising at m/z 193 [C6H9O7]+ and 183 [M-193,C12H23O]+ suggested that a glucuronic acid was esterified with lauric acid. The 1H NMR spectrum of 2 exhibited a one-proton doublets at d 5.26 (J= 7.5 Hz) assigned to anomeric H-1′ proton. The remaining sugar protons resonated as multiplets between d 4.64-3.65. A two-proton multiplet at d 2.29 was due to methylene protons H2-2 adjacent to ester group. The other methylene protons appeared as two-proton multiplets at d 2.02 and 1.58 as a broad singlet at d 1.25 (14H). A three-proton triplet at d 0.87 (J=6.3 Hz) was accounted to terminal C-12 primary methyl protons. Acid hydrolysis of 2 yielded glucuronic acid and lauric acid (co-TLC comparable). On the basis of above discussion the structure of 2 has been elucidated as n-dodecanoyl-O-ß-D-glucuranopyranoside. This is a new acyl glycoside.
CH3OHO1151622cis-Docos-15-enoic acid (1)Compound 3, myristyl-O-ß-D–arabinoside, was obtained as a colorless sticky mass from chloroform-methanol (17:3, v/v) eluants. It gave positive tests for glycosides and displayed characteristic IR absorption bands for hydroxyl groups (3450, 3353 and 3266 cm-1), ester function (1735 cm-1), and long chain aliphatic hydrocarbon (725 cm-1). On the basis of mass spectral data its molecular ion peak was determined at m/z 360 corresponding to the molecular formula of the tridecanyl arabinoside, C19H36O6. The ion peaks generating at m/z 133 [C5H9O4]+, 149 [C5H9O5]+ and 211 [M-149, CH3(CH2)12CO]+ suggested that a C5 sugar unit was esterified with a C14 fatty acid. The 1H NMR spectrum of 3 exhibited a one-proton doublet at d 5.23 (J=7.5 Hz) assigned to ß-oriented anomeric H-1′ protons. Three one-proton multiplets at d 3.85, 3.67 and 3.44 were ascribed to carbinol H-2′, H-3′ and H-4′ protons, respectively. A two-proton doublet at d 3.36 (J=6.8 Hz) was accounted to oxygenated methylene H2-5′ protons. A two-proton triplets at d 2.69 (J=7.9 Hz) was attributed to methylene H2-2 proton adjacent to ester group. The remaining methylene protons appeared as multiplets at d 2.25 and 1.53 and as a broad singlet at d1.25 (18H). A three-proton triplet at d 0.88 (J=6.5 Hz) was accounted to terminal C-13 primary methyl protons. Acid hydrolysis of 3 yielded D-arabinose and myristic acid, co-TLC comparable. On the basis of above spectral data analysis and chemical reactions the structure of 3 has been characterized as n-tetradecanoyl-O-ß-D–arabinopyranoside.
Compound 4, an arachidyl arabinoside, was obtained as a brownish sticky mass from chloroform- methanol (9:1) eluants. It gave positive tests for glycosides and displayed characteristic IR absorption bands for hydroxyl groups (3386 and 3225 cm-1), ester function (1724 cm-1) and long chain aliphatic hydrocarbon (716 cm-1). On the basis of mass spectral data the molecular ion peak of 4 was determined at m/z 444 corresponding to the molecular formula of an acyl glycoside, C25H48O6. The 1H NMR spectrum of 4 exhibited a one-proton doublet at d 5.28 (J=7.8 Hz) assigned to ß-oriented anomeric H-1′ proton. Three one-proton multiplets d 4.29, 4.06 and 3.70 were due to sugar carbinol H-2′, H-3′ and H-4′ protons, respectively. A two-proton doublet at d 3.34 (J=6.9 Hz) was accounted to oxygenated methylene H2-5′ protons. A two-proton triplets at d 2.69 (J=7.5 Hz) was ascribed to methylene H2-2 proton adjacent to ester group. The remaining methylene protons appeared between d 2.23-1.18. A three-proton triplet at d 0.81 (J=6.5 Hz) was accounted for terminal C-20 primary methyl protons. Acid hydrolysis of 4 produced D-arabinose and arachidic acid (co-TLC comparable). On the basis of these evidences the structure of 4 structurally established as n-eicosanoyl-O-ß-D–arabinopyranoside.

CONCLUSION

In the present study characterization of four phytoconstituents from the seeds of L. culinaris have been discussed. This work updated the existing knowledge of this plant. These compounds may be responsible for the quality control purposes and may be use to identifying the authentic samples from adulterated samples.

ACKNOWLEDGEMENTS
The authors are highly thankful to Head, Central Instrumental Facility, Jamia Hamdard, New Delhi, India for recording the spectral data.

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