New fatty acid and acyl glycoside from the aerial parts of Phyllanthus fraternus Webster.

BACKGROUND: Phyllanthus fraternus Webster (Euphorbiaceae) is used to treat dyspepsia, indigestion, jaundice, dysentery, diabetes, influenza, kidney stones, urinary tract diseases, vaginitis, and skin eruptions in traditional systems of medicine.
MATERIALS AND METHODS: The methanol extract of aerial parts of P. fraternus was obtained by soxhlation method. Isolation of compounds was done by silica gel column chromatography. Analytical thin layer chromatography was used to check the homogeneity of eluted fractions. The structures of isolated compounds were established on the basis of spectral studies and chemical reactions.
RESULTS: Phytochemical investigation of a methanolic extract of the aerial parts yielded a new fatty acid characterized as cis-n-octacos-17-enoic acid (5) and a new acyl tetraglycoside formulated as n-dodecanoyl-O-β-D-glucopyranosyl-(2'→1'')-O-β-D-glucopyranosyl-(2''→1''')-O-β-D-glucopyranosyl-(2'''→1'''')-O-β-D-glucopyranoside (7) along with known compounds 1-pentacosanol (1), β-sitosteryl oleate (2), β-sitosteryl linoleate (3), stigmasterol (4) and palmityl glucuronoside (6).

Phyllanthus fraternus Webster (Euphorbiaceae) is a small erect annual herb growing up to 30–60 cm in height and is indigenous to the Amazon rainforest and other tropical areas, including South-Eastern Asia, Western Pakistan, India and China.[12] The plant is astringent, anti-inflammatory, diuretic, cholagog, deobstruent, febrifuge, stomachic and styptic; used to treat jaundice, dyspepsia, indigestion, influenza, diabetes, kidney stones, urinary tract diseases, vaginitis, and skin eruptions. A plant decoction with honey is taken to subside cough and an infusion is drunk as a blood purifier, to reduce blood sugar level and to treat dysentery. The plant is used by mothers after childbirth for painful womb. It has been used in the Ayurvedic preparations such as Chitraka Haritaki, Madhuyastyadi Taila, Pippalyadi Ghrta, Cyavanaprasa and Satavari Guda.[3] The plant is also useful to combat hepatotoxicity, hepatitis B, hyperglycemia and viral and bacterial diseases.[4] Alkamides,[5] seco-sterols, oxygenated heterocyclic compounds, diterpene ester and tetraterpene ketone have been reported from the plant till date.[678] The present work reports the isolation of a new fatty acid and a new acyl tetraglucoside for the first time along with known aliphatic alcohol, palmityl glucuronoside and steroids from the aerial parts of P. fraternus. This study has enhanced the phytochemical nature of the plant. These compounds may be used as a chromatographic marker for standardization, as it is a drug of controversial identity in the traditional systems of medicine.

Materials and Methods

General

Melting points were determined on a perfit apparatus without correction. The infrared (IR) spectra were measured in KBr pellet on a Bio-Rad Fourier transform-IR spectrometer (Spectra Lab Scientific Inc., Ontario, Canada). Ultraviolet (UV) spectra were obtained in methanol with a Lambda Bio 20 spectrometer (Perkin-Elmer, Rotkreuz, Switzerland). 1H (500 MHz) and13C (125 MHz) nuclear magnetic resonance (NMR) spectra were recorded on Bruker spectrospin spectrometer (Bruker AXS, Karlsruhe, Germany). CDCl3 and DMSO-d6(Sigma-Aldrich, Bengaluru, India) were used as solvents and TMS as an internal standard. Electrospray ionization mass spectrometry (ESI MS) analyses were performed on a Waters Q-TOF Premier (Micromass MS Technologies, Manchester, UK) mass spectrometer.

Chromatographic conditions

Column chromatography separation was carried out on silica gel (60–120 mesh, Merck, Mumbai, India). Precoated silica gel plates (Silica gel 60 F254) were used for analytical thin layer chromatography (TLC) to check the homogeneity of fractions. The spots were visualized by exposure to iodine vapors and UV radiations.

Plant material

The aerial parts of P. fraternus was collected from the herbal garden of Jamia Hamdard, New Delhi and identified by Dr. H.B. Singh, Head, Raw Materials Herbarium and Museum, National Institute of Science Communication and Information Resources (NISCAIR), New Delhi. A voucher specimen of the drug was deposited in the herbarium of NISCAIR with a reference number NISCAIR/RHMD/Consult/2011-12/1756/56.

Extraction and isolation

The air dried coarsely powdered aerial parts of P. fraternus (500 g) were extracted with methanol using a Soxhlet apparatus for 18 h. The extract was concentrated in vacuum to yield a brown semisolid mass (109 g). A small portion of the extract was analyzed chemically to determine the presence of different chemical constituents. The extract (100 g) was suspended in distilled water (500 ml) and partitioned with petroleum ether (500 ml × 5), successively to give petroleum ether (5.31 g) and water soluble (94.69 g) fractions. The petroleum ether soluble fraction was subjected to silica gel column chromatography (column A) eluting with petroleum ether-chloroform (1:3) to obtain compound 1 and 2 [Figure 1]. Analytical TLC was used to check the homogeneity of eluted fractions. The aqueous fraction was dried using rotary evaporator under reduced pressure (72 mbar) at 50°C and dissolved in minimum amount of methanol to adsorbed on silica gel (60–120 mesh) for preparation of slurry. The slurry was dried and subjected to silica gel column loaded in chloroform (column B). The column was eluted with chloroform and chloroform-methanol (99:1, 19:1 and 3:1) mixtures to obtain compound 3–7 [Figure 1].

Figure 1

Structure of compounds 1–7 isolated from the methanolic extract of aerial parts of Phyllanthus fraternus

1-Pentacosanol (1)

Elution of the column A with petroleum ether-chloroform (1:3) gave a colorless powder of 1, 960 mg (0.96% yield); Rf0.45 (petroleum ether-choloroform, 1:1); mp 70–71°C; IR νmax (KBr) cm−1: 3301 (O-H), 2915 (C-H), 2846 (C-H), 1463 (CH2), 722 (aliphatic chain); 1H NMR (CDCl3): δ 3.57 (2H, t, J = 6.6 Hz, CH2-1), 1.49 (2H, m, CH2), 1.18 (38H, brs, 19 × CH2), 0.81 (3H, t, J = 6.5 Hz, Me-25);13 C NMR (CDCl3): δ 63.21 (C-1), 32.93-22.69 (23 × CH2), 14.31 (Me-25); ESI MS m/z (rel. int.): 368 [M]+ (C25H52O) (41.8).

β-Sitosteryl oleate (2)

Further elution of the column A with petroleum ether-chloroform (1:3) furnished a pale yellow semisolid mass of 2, 725 mg (0.725% yield); Rf0.65 (petroleum ether-chloroform, 1:3); m.p: 106–108°C; IR νmax (KBr) cm−1: 1722 (O = C-O, ester), 1635 (C = C), 1435 (CH2), 1364 (CH3), 1240 (=C-H), 1062 (C-H, Ar), 719 (aliphatic chain); 1H NMR (CDCl3): δ 5.33 (1H, m, H-6), 5.28 (1H, m, H-9’), 5.05 (1H, m, H-10’), 4.08 (1H, brm, w1/2 = 18.5 Hz, H-3α), 2.23 (2H, t, J = 4.8 Hz, H2-2’), 0.99 (3H, brs, Me-19), 0.90 (3H, d, J = 6.3 Hz, Me-21), 0.87 (3H, d, J = 6.5 Hz, Me-26), 0.84 (3H, d, J = 6.1 Hz, Me-27), 0.82 (3H, t, J = 6.6 Hz, Me-18’), 0.80 (3H, d, J = 6.0 Hz Me-29), 0.67 (3H, brs, Me-18), 2.79–1.28 (31H, m, 12 × CH2,7 × CH), 1.24 (22H, brs, 11 × CH2);13 C NMR (CDCl3): δ 67.14 (C-3), 42.31 (C-4), 138.27 (C-5), 120.63 (C-6), 12.05 (C-18), 19.42 (C-19), 18.83 (C-21), 45.81 (C-24), 29.09 (C-25), 19.88 (C-26), 19.20 (C-27), 11.90 (C-29), 173.25 (C-1’), 37.69–22.68 (23 × CH2), 14.17 (C-18’); ESI MS m/z (rel. int.): 678 [M]+ (C47H82O2) (2.3), 413 (62.1), 282 (51.4).

β-Sitosteryl linoleate (3)

Elution of the column B with chloroform afforded a pale yellow semisolid mass of 3, 408 mg (0.40% yield); Rf0.63 (petroleum ether-chloroform, 1:3), mp: 117–118°C; IR νmax (KBr) cm−1: 1721 (O = C-O, ester), 1645 (C = C), 1445 (CH2), 1364 (CH3), 1243 (=C-H), 1038 (C-H, Ar), 717 (aliphatic chain); 1H NMR (CDCl3): δ 5.31 (1H, m, H-6), 5.19 (1H, m, H-9’), 5.01 (1H, m, H-10’), 4.94 (1H, m, H-12’), 4.90 (1H, m, H-13’), 4.09 (1H, brm, w1/2 = 18.1 Hz, H-3α), 2.24 (2H, t, J = 7.3 Hz, H2-2’), 0.94 (3H, brs, Me-19), 0.90 (3H, d, J = 6.3 Hz, Me-21), 0.85 (3H, d, J = 6.5 Hz, Me-26), 0.81 (3H, d, J = 6.1 Hz, Me-27), 0.77 (3H, t, J = 6.6 Hz, Me-18’), 0.74 (3H, d, J = 6.0 Hz, Me-29), 0.61 (3H, brs, Me-18);13 C NMR (CDCl3): δ 71.83 (C-3), 140.76 (C-5), 121.70 (C-6), 11.87 (C-18), 19.42 (C-19), 18.83 (C-21), 19.88 (C-26), 19.20 (C-27), 11.99 (C-29), 173.32 (C-1’), 139.26 (C-9’), 130.92 (C-10’), 35.58 (C-11’), 128.85 (C-12’), 114.01 (C-13’), 14.15 (C-18’); ESI MS m/z (rel. int.): 676 [M]+ (C47H80O2) (2.3), 413 (22.1), 280 (15.3).

Stigmasterol (4)

Further elution of the column B with chloroform yielded a colorless amorphous powder of 4, 120 mg (0.12% yield), Rf0.54 (chloroform); mp 168–170°C; IR νmax (KBr) cm−1: 3430 (O-H), 1641 (C = C); 1H NMR (CDCl3): δ 5.33 (1H, d, J = 3.6 Hz, H-6), 5.20 (1H, m, H-22), 5.15 (1H, m, H-23), 3.45 (1H, brm, w1/2 = 16.5 Hz, H-3α), 0.94 (3H, brs, Me-19), 0.86 (3H, d, J = 6.4 Hz, Me-21), 0.79 (3H, d, J = 6.4 Hz, Me-26), 0.75 (3H, d, J = 7.6. Hz, Me-27), 0.73 (3H, d, J = 6.8 Hz, Me-29), 0.61 (3H, brs, Me-18);13 C NMR (CDCl3): δ 71.81 (C-3), 56.87 (C-14), 55.96 (C-17), 11.99 (C-18), 19.41 (C-19), 18.79 (C-21), 138.13 (C-22), 129.22 (C-23), 45.83(C-24), 27.28 (C-25), 19.83 (C-26), 18.99 (C-27), 23.11 (C-28), 11.87 (C-29); ESI MS m/z (rel. int.): 412 [M]+ (C29H48O) (18.2).

n-Octacosenoic acid (5)

Elution of the column B with chloroform-methanol (99:1) furnished cream-colored flakes of 5, recrystallized from chloroform, 96 mg (0.096% yield); Rf0.64 (chloroform-methanol, 9.5:0.5); mp 96–98°C; UV λmax (MeOH): 208 (log ε 3.7); IR νmax (KBr) cm−1: 3360 (O-H, carboxylic), 2903 (C-H), 2837 (C-H), 1701 (C = O, carboxylic), 1633 (C = C), 1457 (CH2), 1377 (CH3), 1281 (=C-H), 718 (aliphatic chain); 1H NMR (CDCl3): δ 5.14 (1H, m, w1/2 = 8.1 Hz, H-17), 5.07 (1H, m, w1/2 = 8.5 Hz, H-18), 2.27 (2H, t, J = 7.2 Hz, H2-2), 2.02 (2H, m, H2-16), 1.97 (2H, m, H2-19), 1.54 (2H, m, H2-3), 1.18 (30H, brs, 15 × CH2), 1.11 (10H, brs, 5 × CH2), 0.81 (3H, t, J = 6.4 Hz, Me-28);13 C NMR (CDCl3): δ 178.50 (C-1), 139.20 (C-17), 114.07 (C-18), 33.81 (CH2), 31.94 (CH2), 29.31 (12 × CH2), 29.52 (3 × CH2), 29.45 (CH2), 29.37 (CH2), 27.26 (CH2), 29.13 (CH2), 29.06 (CH2), 24.71 (CH2), 22.70 (CH2), 14.11 (Me-28); ESI-MS m/z (rel. int.): 422 [M]+ (C28H54O2) (69.4), 281 (28.9), 255 (72.6).

Palmityl glucuronoside (6)

Elution of the column B with chloroform-methanol (19:1) gave a cream-colored sticky mass of 6, 63 mg (0.063% yield); Rf: 0.48 (chloroform-methanol, 99:1); mp 138–140°C; IR νmax (KBr) cm−1: 3446 (O-H), 3250 (O-H, carboxylic), 1722 (O = C-O, ester), 1695 (O-H, carboxylic), 729 (aliphatic chain); 1H NMR (DMSO-d6): δ 5.85 (1H, d, J = 7.1 Hz, H-1’), 4.40 (1H, m, H-5’), 4.22 (1H, m, H-2’), 4.07 (1H, m, H-4’), 3.64 (1H, m, H-3’), 2.28 (2H, t, J = 7.6 Hz, H2-2), 1.53 (2H, m, CH2), 1.22 (6H, brs, 3 × CH2), 1.18 (18H, brs, 9 × CH2), 0.85 (3H, t, J = 6.4 Hz, Me-16);13 C NMR (DMSO-d6): δ 172.81 (C-1), 101.03 (C-1’), 71.81 (C-2’), 71.16 (C-3’), 69.55 (C-4’), 75.19 (C-5’), 179.29 (C-6’), 14.15 (C-16); ESI MS m/z (rel. int.): 432 [M]+ (C22H40O8) (2.5), 255 (100), 239 (11.9), 193 (33.1).

Lauryl tetraglucoside (7)

Elution of the column with chloroform-methanol (3:1) furnished a yellow powder of 7, recrystallized from chloroform-methanol (1:1), 81 mg (0.081% yield), Rf0.53 chloroform-methanol-acetic acid (3:1:0.02); mp 133–134°C; UV λmax (MeOH): 250 nm (log ε 3.6); IR νmax (KBr) cm−1: 3506 (O-H), 3441 (O-H), 3348 (O-H), 3225 (O-H), 2906 (C-H), 2841 (C-H), 1723 (O = C-O, ester), 1445 (CH2), 1372 (CH3); 1H NMR (DMSO-d6): δ 5.32 (1H, d, J = 7.2 Hz, H-1’), 5.08 (1H, d, J = 7.5 Hz, H-1’’), 4.99 (1H, d, J = 7.1 Hz, H-1’’’), 4.93 (1H, d, J = 7.3 Hz, H-1’’’’), 4.39 (1H, m, H-5’), 4.30 (2H, m, H-5’’, H-5’’’), 4.16 (1H, m, H-5’’’’), 3.79 (1H, m, H-2’), 3.73 (2H, m, H-2’’, H-2’’’), 3.61 (1H, m, H-2’’’’), 3.52 (3H, m, H-3’, H-3’’, H-3’’’), 3.49 (1H, m, H-3’’’’), 3.46 (3H, m, H-4’, H-4’’, H-4’’’), 3.37 (1H, m, H-4’’’’), 3.18 (2H, brs, H2-6’), 3.09 (2H, brs, H2-6’’), 3.05 (2H, brs, H2-6’’’), 3.02 (2H, brs, H2-6’’’’), 2.23 (2H, t, J = 7.3 Hz, H2-2), 2.10 (2H, m, CH2), 1.96 (2H, m, CH2), 1.47 (2H, m, CH2), 1.19 (12H, brs, 6 × CH2), 0.78 (3H, t, J = 6.3 Hz, Me-12);13 C NMR (DMSO-d6): δ 174.96 (C-1), 55.39 (C-2), 31.82 (C-3), 29.49 (C-4), 29.49 (C-5), 29.49 (C-6), 29.16 (C-7), 29.46 (C-8), 29.15 (C-9), 25.08 (C-10), 22.55 (C-11), 14.40 (C-12), 104.63 (C-1’), 83.39 (C-2’), 72.02 (C-3’), 68.28 (C-4’), 77.21 (C-5’), 61.61 (C-6’), 102.45 (C-1’’), 82.37 (C-2’’), 70.78 (C-3’’), 64.53 (C-4’’), 76.34 (C-5’’), 61.56 (C-6’’), 96.51 (C-1’’’), 81.41 (C-2’’’), 70.36 (C-3’’’), 64.20 (C-4’’’), 75.81 (C-5’’’), 61.51 (C-6’’’), 92.30 (C-1’’’’), 75.30 (C-2’’’’), 69.45 (C-3’’’’), 63.42 (C-4’’’’), 76.21 (C-5’’’’), 61.48 (C-6’’’’); ESI MS m/z (rel. ret.): 848 [M]+ (C36H64O22) (1.1), 685 (7.2), 669 (15.2), 665 (3.6), 649 (6.3), 327 (27.8), 199 (18.5), 183 (14.6), 179 (12.3), 163 (7.4).

Results and Discussion

Compounds 1–4 and 6 were the known phytoconstituents characterized as 1-pentacosanol,[910] β-sitosteryl oleate,[11] β-sitosteryl linoleate,[12] stigmasterol[13] and palmityl glucuronoside,[14] respectively [Figure 1].

Compound 5, named n-octacosenoic acid, was obtained as cream-colored flakes from chloroform-methanol (99:1) eluants. It gave effervescence with sodium bicarbonate indicating the carboxylic nature of the compound. Its IR spectrum showed characteristic absorption bands for the carboxylic group (3360, 1701 cm−1), unsaturation (1633 cm−1) and long aliphatic chain (718 cm−1). On the basis of mass and13 C NMR spectra of 5, its molecular ion peak was established at m/z 422 corresponding to a molecular formula of a long chain unsaturated fatty acid, C28H54O2. It indicated the presence of two double bond equivalents which were adjusted one each in a vinylic linkage and the carboxylic group. The ion peaks arising at m/z 255 ([CH2]15 COOH+) and 281 (CH = CH[CH2]15 COOH+) indicated the existence of the vinylic linkage at C-17 carbon. The 1H NMR spectrum of 5 displayed two one-proton multiplets at δ 5.14 (w1/2 = 8.1 Hz), 5.07 (w1/2 = 8.5 Hz) assigned to cis-oriented vinylic H-17 and H-18 protons, respectively. A two-proton triplet at δ 2.27 (J = 7.2 Hz) was ascribed to methylene H2-2 protons adjacent to the carboxylic group. The other methylene protons appeared between δ 2.02 and δ 1.11. A three-proton triplet at δ 0.81 (J = 6.4 Hz) was accounted to the primary methyl C-28 protons. The13 C NMR spectrum of 5 exhibited important signals for carboxylic carbon at δ 178.50 (C-1), vinylic carbons at δ 139.20 (C-17), and 114.07 (C-18), methyl carbon at δ 14.11 (C-28) and methylene carbons between δ 33.81 and δ 22.70. On the basis of spectral data analysis and chemical reactions, the structure of 5 has been elucidated as as cis-n-octacos-17-enoic acid [Figure 1]. This is a new fatty acid reported for the first time from this plant.

Compound 7, designated as lauryl tetraglucoside, was obtained as a yellow powder from chloroform-methanol (3:1) eluants. It gave positive tests of glycosides and showed IR absorption bands for hydroxyl groups (3506, 3441, 3348, 3225 cm−1) and ester group (1723 cm−1). Its molecular ion peak was determined at m/z 848 on the basis of mass and13 C NMR spectra which corresponded to the molecular formula of an acyl tetraglycoside (C36H64O22). The ion peaks generating at m/z 183 [CH3(CH2)10 CO]+, 199 [CH3(CH2)10 COO]+, 665 (M-183)+ and 649 (M-199)+ suggested that lauryl group was attached to a tetraglycoside chain. The ion fragments arising at m/z 179 [C6H11O6]+, 669 (M-179)+, 685 (M-163)+ and 325 [C6H11O6-C6H10O3]+ indicated that C6-sugar units were linked in the glycosidic chain. The 1H NMR spectrum of 7 displayed four one-proton doublets at δ 5.32 (J = 7.2 Hz), 5.08 (J = 7.5 Hz), 4.99 (J = 7.1 Hz) and 4.93 (J = 7.3 Hz) assigned correspondingly to anomeric H-1’, H-1’’, H-1’’’ and H-1’’’’ protons. The other sugar protons appeared between δ 4.39 and δ 3.02. A two-proton triplet at δ 2.23 (J = 7.3 Hz) was ascribed to methylene H2-2 protons nearby to the ester group. The other methylene protons resonated from δ 2.10 to δ 1.19. A three-proton triplet at δ 0.78 (J = 6.3 Hz) was accounted to primary C-12 methyl protons. The13 C NMR spectrum of 7 exhibited signals for ester carbon at δ 174.96 (C-1), anomeric carbons at δ 104.63 (C-1’), 102.45 (C-1’’), 96.51 (C-1’’’) and 92.30 (C-1’’’’), other sugar carbons between δ 83.39 and δ 61.48, methylene carbons from δ 55.39 to δ 22.55 and methyl carbon at δ 14.40 (C-12). The presence of carbon signals in the deshielded region at δ 83.39 (C-2’), 82.37 (C-2’’) and 81.41 (C-2’’’) in the13 C NMR spectrum suggested (2→1) linkage of the sugar units. Acid hydrolysis of 7 yielded lauric acid and D-glucose (co-TLC comparable). On the basis of this discussion the structure of 7 has been established as n-dodecanoyl-O-β-D-glucopyranosyl-(2′→1′)-O-β-D-glucopyranosyl- (2′→1‴)-O-β-D-glucopyranosyl-(2‴→1′′)-O-β-D-glucopyranoside [Figure 1]. This is a new acyl tetraglycoside reported for the first time from this plant.

Conclusion

The present work reports the isolation of a new fatty acid, and acyl tetraglucoside along with known aliphatic alcohol, palmityl glucuronoside and steroids from the aerial parts of P. fraternus. This study has enhanced the phytochemical nature of the plant. These compounds may be used as a chromatographic marker for standardization, as it is a drug of controversial identity in the traditional systems of medicine.

Financial support and sponsorship

Department of Science and Technology, New Delhi, Government of India.

Conflicts of interest

There are no conflicts of interest.