Phenolics and alkaloids from the fruits of piper longum l

Journal o f Medicinal Materials, 2022, Vol 27, No (pp 90 - 94) PHENOLICS AND ALKALOIDS FROM THE FRUITS OF PIPER LONG UM L Nguyên Tra My1, Nguyên Thi Thu1, Vu Thi Diep1, Hoang Thi Dieu Huong1, Nguyên Thi Hang1, Nguyên Thi My Linh2, Vu Duc Loi2, Do Thi Ha1’* 1National Institute o f Medicinal Materials (NIMM), Hanoi, Vietnam; 2VNU Unỉversity o f Medicine and Pharmacy, Hanoi, Vietnam *Coưesponding author: hado.nimms@gmail.com (Received April 05th, 2022) Summary Phenolỉcs and Alkaloids from the Fruits of Pỉper longum L Seven compounds, including avenalumic acid methyl ester (1), methyl piperate (2), piperine (3), pipemonaline (4), (2£',42,8£')-,V-[9-(3,4-methylenedioxyphenyl)-2,4,8-nonatnenoy]] piperidine (5), pipercide (6), and gúineensine (7) weré ỉsolated fróm the ethyl acetate extract of Piper longum L fruits The structures óf thêse compounds were identified by using extensive nuclear mãgnetic resonance spectroscopý (NMR) data, mass spectrometry (MS), and comparing with published literature Compounds and were the ốrst reporí írom this species Keytvords: Piper longum, Fruìt, Phenolics, Alkaloids, Piperine Introduction Piper ỉongum L., a species of Piperaceae [1], distributes mainly in India, South China, Laos, Vietnam, and some other counừies in Southeast Asia In Vietnam, p ỉongum grows in mountainous and midland provinces, especially those with limestone mountains [2] According to traditional medicine, p longum 1S hot, pungent, and is often used to treat respiratory diseases such as sinusitis, pain in the nostriĩs, and nasal cavity and digestive diseases such as abdominal pain, cold stomach causing vomiting, diarrhea, indigestion [1],[2] Studies on Chemical composition m the p ĩongum fruits showed a large number of alkaloids and relăted compounds [3],[4] In addition, p longum contains lignans, essentìal oils, and organic acids [3],[4] Bĩological investigation of extrăcts and compounds from p longum ĩruits showed anti-cancer [5],[6], immimomodulation [7], antìoxidant [8], hepatoprotection [8],[9], anti-inílammatìon 90 [10],[11], and antibacterial eữects [12] Piperine, MI alkaloid foimd in plants such as p nigrum and p longum, shows anti-lung cancer and metastatic actmty Piperlonguminin inhibits a-MSH-induced tyrosmase synthesis [5],[6] Besides, p ỉongum extracts and piperinic acid decreased dosedependently lympho cell (CD4+ and CD8+cells) and cytokine levels in Baìb/C mice [7] To our lóiowledge, until now no report about the Chemical composition of this species collected in Vietnam has been published In thỉs paper, we report the isolation and stmctural elucidation of phenolics and alkaloids compounds írom the fruits of p longum Materials and methods 2.1 Pỉant materỉal Samples of Piper longum L., Piperaceae were collected in Dak Song, Dak Nong, Vietnam in September 2020 The plant was identiíied by Assoc Prof Pham Thanh Huyen and MSc Nguyên Van Hieu (National Institúte of Medicinal Materials, Lournal ofMedicinalMaterials, 2022, Vol 27, No NIMM) A voucher specimen (DL-200420) was deposited in the Department of Medicinal Material Resources, NIMM (Hanoi, Vietnam) 2.2 General experimentaỉ procedures N M R spectra were recorded on a Bruker Avance Digital 500 MHz N M R specữometer (Karlsruhe, Germany) using TMS as the intemal Standard, J in Hz, at 294 K ESI-MS data were analyzed vía a Q Exactive Orbitrap Mass Spectrometer Silica gel (Merck, 63-200 mm particìe size), and YMC gel (75-150 pm) were used for column chromatography (CC) Thin-layer chromatography (TLC) was carried out with silica geỉ 60F 254 and R P - 18F 254 plates Spots were detected under v or by sprayúig with 10% sulíuric acid in 96% ethanol (EtOH) and heating 2.3 Extraction and isoỉation The dried lruits of p longum (3.0 kg) were extracted and retluxed with 70% EtOH (3 times, hours each time) at 80°c The extracts were íĩltered, combined, and evaporated under reduced pressure, yielding a green residue (600.33 g), which was suspended in water and successively separated with n-hexane and ethyl acetate (EtOẤc) The EtOAc extract (192.32 g) was separated on a siỉica geỉ cc with a gradient mixture of H-hexane-EtOAc (100:00:100, v/v) of increasing polarity as eluents to give ten tractions (1A-1K) and three compounds (1.44 g), (24.75 g), and (0.48 g) Fractíon II (5.03 g) was ữactionated on RP-C18 gel c c eluting with an isocratic mixture of methanol-water (4:1, v/v) to obtain nine fractions (2A-2I) and two compounds (0.86 g) and (0.12 g) Fraction 2D (2.02 g) was applied to sìlica gel c c with an isocratic elution of n-hexane-EtOAc (4:1, v/v) to ĩịrd three fractions (3A-3C) Compound (10 mg) was obtained from ìraction 3C (56 mg) by ùsing silica gel c c with an elution mixture of ũ-hexane-acetone (5:1, v/v) Fraction 1G (2.02 g) was applied to RP-C18 gel c c with an isocratic elution of mẽthanol-water (3:2, v/v) to afford six ữactions (6A-6F) Purification of ữaction 6D (60.0 mg) by RP-C18 gel c c with a mixture of acetone-H20 (3:2, v/v) to isolate compound (10 mg) Compound 1: Pale yellow powder; 'H-NMR (600 MHz, CD3OD) Ổn 5.97 (1H, d, = 15.0 Hz, H2), 7.45 (1H, dd, 7= 10.8, 15.0 Hz, H-3), 6.84 (1H, dd, 7= 10.8,15.0 Hz, HA), 6.93 (1H, d ,7 = 15.0 Hz, H-5), 7.40 (2H, d, J = 8.4 Hz, H-2', H-6'), 6.80 (2H, d = 8.4 Hz, H-3', H-5'), 3.76 (3H, s, ÓCH3); 13Ó NMR (125 MHz, CD3ÓD) ỏc: 169.6 (C-l), 119.6 (C-2), 147.4 (0 ), 124.4 (0 ), 142.5 (0 ), 129.1 (0 , 130.1 ( , , 116.6 (C-3', '), 160.0 (CA'), 51.9 (OCH3) Compound 2: White powder; 'H-NMR (600 MHz, CDCI3) ỔH: 5.95 (1H, d, J = 15.6 Hz, H-2), 7.42 (1H, dd, ỹ = 11.4, 15.6 Hz, H-3), 6.70 (1H, dd, J = 11.4,15.0 Hz, H-4), 6.81 (1H, d, J= 15.0 Hz, H-5), 6.99 (1H, d, J = 1.8 Hz, H-2% 6.78 (1H, d, J = 8.4 Hz, H-5'), 6.91 (1H, dd, J = 1.8,8.4 Hz, H-6'), 5.98 (2H, s, OCH2O), 3.76 (3H, s, OCH3); 13C-NMR (150 MHz, CDCI3) Sc: 167.6 (C-l), 123.0 (C-2), 145.0 (C-3), 124.5 (C-4), 140.3 (0 ), 130.6 (c-ỉ% 105.9 (020,148.6 (030,148.3 (040,108.5 (050, 120.0 (0 , 101.4 (OCH2O), 51.5 (OCH3); ESÍMS: m/z 233.10 [M+H]+ Compound 3: Pale yellow powder; 'TỈ-NMR (600 MHz, CDCI3) ỏn 6.44 (1H, d, J = 15.0 Hz, H2), 7.40 (1H, m, H-3), 6.73 (2H, m, HA, H-5), 6.98 (ÍH, d, = Hz, H-20,6.78 (ÍH, d, J = 7.8 Hz, H50,6.89 (1H, dd, J= 1.8, 7.8 Hz, H-60, 5.97 (2H, s, OCH2O), 3.58 (4H, br s, H-2", H-6'O, 1.58 (4H, m, H-3", H-5'O, 1.66 (2H, m, H-4'0; l3C-NMR (150 MHz, CDCÍ3) ơc: 165.5 (C-l), 120.1 (C-2), 142.5 (0 ), 125.4 (0 ), 138.2 (0 ), 131.1 (0 , 105.7 (020,148.2 (030,148.1 (CAO, 108.5 (050,122.5 (0 , 101.3 (OCH2O), 43.3 (C-2'O, 26.7 (C-3'O, 24.7 (CA'0, 25.6 (C-5'O, 46.9 (C-6'0; ESI-MS: m/z 286.2 [M+H]+ Compound 4: White powder; 'H-NMR (500 MHz, CDCls) ỎH 6.22 (1H, d, J = 15.0 Hz, H-2), 6.82 (1H, dt, >= 15.0,7.0 Hz, H-3), 2.20 (4H, m, H4, H-7), 1.67 (4H, m, H-5, H-6), 6.02 (1H, m, H-8), 6.27 (ÍH, d, = 15.5 Hz, H-9), 6.88 (1H, d, = 1.5 Hz, H-20, 6.74 (2H, m, H-5', H-60, 3.48 (2H, br s, H-2'0,1.67 (6H, m, H-3", HA", H-5'O, 3.59 (2H, br s, H-6'0,5.92 (2H, s, OCH2O); 13C-NMR (125 MHz, CDCI3) ỏc: 165.6 (C-l), 120,5 (C-2), 145.6 (C-3), 32.7 (C-4), 29.0 (0 ), 29.7 (C-6), 32.4 (C-7), 128.9 (C-8), 129.6 (0 ), 132.4 (C-10,105.4 (C-20,146.6 (030,147.9(040,108.2 (050,120.2 (060.100.9 (OCH2O), 43.1 (C-2'0,25.6 (C-3'0,24.7 (CA'0,26.6 (C-5'0,46.9 (C-6'0; ESI-MS: m/z 343.3 [MAỈ]+ Compound 5: White powder; 'H-NMR (500 MHz, CDCI3) ỏ h : 6.34 (1H, d, 7= 15.0 Hz, H-2), 7.58 (1H, dd, = 11.5,14.5 Hz, H-3), 6.16 (1H, t, = 11.5 Hz, H-4), 5.78 (ÍH, dt, = 11.0, 8.0 Hz, H-5), 2.46 (2H, m, H-6), 2.29 (2H, m, H-7), 6.02 (1H, dt, = 16.0, 7.0 Hz, H-8), 6.30 (1H, d, = 16.0 Hz, H-9), 6.87 (1H, s, H-20, 6.74 (2H, m, H-5', H-6Ó, 5.93 (2H, s, OCH2O), 3.49 (2H, br s, H-2"), 1.62 (6H, m, H-3", HA", H-5'O, 3.62 (2H, br s, H-6"); l3C-NMR (125 MHz, CDCÍ3) ỗc: 165.6 (Ĩ l), 121.1 (C-2), 137.1 (0 ), 127.5 (C-4), 138.2 (C-5), 27.9 (C-6), 32.7 (0 ), 127.8 (C-8), 130.2 (C-9), 132.2 (0 , 105.5 (020,146.7(030,147.9 (CAO, 108.2 (0 , 120.4 (0 , 100.9 (OCH2O), 43.2 (02"), 25.6 (O 3'0, 24.6 (CA'0, 26.7 (C-5'O, 46.9 (C-6'O; ESI-MS: mà 339.2 [M-H]- _ Compound : White powder; 'H-NMR (500 MHz, CD3OD) Ỗh: 5.95 (1H, d, = 15.0 Hz, H-2), 7.13 (1H, d d ,7 = 11.0,15.0 Hz, H-3), 6.22 (ÍH, dd, = 11.0,15.0 Hz, HA), 1.51 (4H, m, H-7, H-8), 2.20 (4H, m, H 6, H-9), 6.10 (2H, m, H-5, H-10), 6.31 lournal o f MedicinalMaterials, 2022, Voi 27, No 91 OliH, đ, J = 16.0 Hz, H-l 1), 6.91 (1H, d, J = 2.0 Hz, IH-2% 6.73 (1H, d, J = 8.0 Hz, H-50, 6.77 (1H, dd, i - ‘1.5,8.0 Hz, H-60, 5.92 (2H, s, 0CH 20), 3.08 (2H, d , J = 7.0 Hz, H-1'O, 1.81 (1H, m, H-2'O, 0.94 (6H, d, J = 6.5 Hz, H -3, H-4'O; 13ONMR (125 MHz, CD3OD) ỗc: 169.2 (C-l), 123.1 (0 ), 142.1 (0 ), 129.7 (C-4), 143.8 (0 ), 33.8 (0 ), 30.1 (0 ), 29.7 (0 ), 33.7 (0 ), 129.9(010), 131.0 (011), 133.8(01'), 106.3 (0 , 149.4 (03% 148.1 (C40, 109.0(050, 121.3 (0 , 102.2 (OCH2O), 48,0 (C1'0, 29.5 (C-2'O, 20.5 (0 ", O4'0; ẼSÍ-MS: w/z 356.3 [M+H]+ m Compound 7: Pale yellow powder; 'H-NMR (600MHz, CDCI3) Ỗh: 5.75 (1H, d ,J = 16.2 Hz, H2), 7.18 (1H, dd, J= 10.8, 15.0 Hz, H-3), 6.12 (1H, d d,J= 10.8,15.0 Hz, H-4), 6.08 (2H, m, H-5, H-l 2), 2.15 (4H, m, 11-6,11-11), 1.37 (8H, m, H-7, H-8, H9, H-10), 6.28 (1H, d, J = 15.6, H-13), 6.88 (1H, d, J= 1.2 Hz, H-2% 6.74 (2H, m, H-5', H-6% 5.92 (2H, s, OCH2O), 5.55 (1H, s, NH), 3.16 (2H, t, J = 13.2 Hz, H-l"), 1.76 (1H, m, ĩỉ-2,'% 0.92 (6H, d, J = 6.6 Hz, H-3", H-4"), 13C-NMR (150 MHz, CDCI3) ỏó 166.4 (C-l), 121.8 (C-2), 141.3 (C-3), 128.3 (0 ), 143.0 (0 ), 32.9 (0 ), 29.4 (C-7), 29.0 (C-8, C-9), 28.6 (010), 32.8 (C-lí), 129-4 (0 ,0 ), 132.5 ( O l i 105.4 (02% 147.9 (C-3% 146.6 (CA% 108.2 (050,120.2 (C-6 Ó, 100.9 (OCH2O), 47.0 (C-1'O, 28.7 (C-2'O, 20.1 (C-3", 04 "); ESI-MS: m/z 382.3 [M-ÍỊ]- Results and discussion Seven compounds were isolated from the 70% ethanol extract of the fruits of p longum (1-7) (Fig 1) From this extract, the identities of compounds wete verified by comparing theừ physicochemical and spectroscopic data to published values cômpound was isolated as pale-yellow powder The 'H-NMR of showed signals õf a phydroxybenzene structure at ỗH 7.40 (2H, d , J = 8.4 Hz, H-2', H-60 and 6.80 (2H, d, J = 8.4 Hz, H-3', H50- Besides, showed sịgnals of two contiguous /rans-double bonds at Ổh ;5:97 (1H, d, J = 15.0 Hz, H-2), 7.45 (1H, dd, J= 10.8,15.0 Hz, H-3), 6.84 (1H, dd, j = 10.8,15.0Hz, H-4) and6.93 (1H, d , / = 15.0 Hz, H-5) and a methoxy group at ổH 3.76 (3H, s, OCH3) The 13ONMR showed signals of 12 carbon including two signals of the /?-hydroxybenzene skeleton [ổc 130.1