Introduction

Herbal medicines were used throughout human history and played a crucial role for the treatment of a wide variety of diseases and ailments. Ancient, Indian medicinal systems viz. Ayurveda, Siddha, Unani etc. and native health traditions provided a robust base for the use of medicinal plants in the management and treatment of various illness (Kayser, Kidderlen, & Croft, 2000). Herbal medication was additionally an efficient healing technique but was viewed less sky-high (Jayaprasad, Thamayandhi, & Sharavanan, 2012). Herbal products were discarded from typical medicinal use, not essentially as a result of they were ineffective (Anbazhakan & Balu, 2007) but due to the reason that they weren't as economically profitable as the newer allopathic medications (Nagori et al., 2011). But now a day, the interest in "natural health" and therefore, the use of medicinal plants accrued steadily (Suresh et al., 2011). Premna latifolia, small bushy and thick trees, belonging to the Verbenaceae family (Yoganarasimhan, 2000) are spread across various Indian states like Kerala, Bihar, Karnataka, West Bengal, Assam etc. (Fletcher, 1938). In vernacular languages, it is known as Bakar (Hindi), Aginimantha (Sanskrit), Knappa (Malayalam) and Erumaimunnai (Tamil) (Aravindakshan & Bai, 1996; Krishnamurthi, 2005; Moldenke, 1983; Nadkarni, 1985). Premna latifolia is used as cariotonic, hepatoprotective, diuretic and antipyretic. Currently, two novel icetexanediterpenes and two new glycosides have been isolated from Premna latifolia (Ghosh, Das, & Dinda, 2014; Thirumalai, Paridhavi, & Gowtham, 2013). As a part of continued efforts directed towards the study of the biologically active compounds from the various medicinal herbal plants, this work was designed and executed to isolate and characterize compounds from Premna latifolia roots (Wagner & Bladt, 1996).

Materials and Methods

Procurement and authentication of roots of Premna latifolia

Roots of Premna latifolia was purchased from a local market in Kasargod, Kerala, India and authenticated by NISCAIR New Delhi, India. [Authentication number–NISCAIR/RHMD/2011-12/1922/222/01]

Extraction and isolation of compounds from dried roots

The coarsely pulverised dried roots of Premna latifolia (5kg) were subjected to soxhlet extraction with absolute ethyl alcohol (50 liters) (Silverstein, Webster, & Kiemle, 2005). The concentrated extract was later subjected to gradient elution by using column chromatography where, methanol, chloroform, ether were used in variable proportions (Brain & T.D.Turner, 1985). The fractions of the plant were collected on an individual basis and combined based on TLC pattern (Ali, 2008; Harbone, 1998). The individual compounds were further separated and sublimated using preparative TLC and Toluene: ethyl acetate (9.3:0.7) as mobile phase (Gupta, Tandon, & Sharma, 2008). The isolated compounds were characterized using FTIR, LC-MS, 1H NMR, 13C NMR and 2D-NMR techniques (Kamalambigeswari, Yadav, Sivaswamy, & Ushani, 2018; Parameswari, Aswini, Aruna, & Reddy, 2015).

Compound 1: n-Tridecanyl n-Tetracosanoate

When the column was eluted with chloroform 100%,it resulted in a reddish brown solid mass (Asharani, Thirumalai, Paridhavi, & Gowtham, 2013; Gowtham, Asharani, & Paridhavi, 2018) of compound 1 of yield 1.352 g (0.233% yield) with R_f value 0.62 (Toluene: Ethylacetate (9.3:0.7)); m.p.57-59°C; IR λ max (KBr): 2926, 2855, 1740, 1632, 1461, 1377, 1250, 1197,1172, 757cm^-1 (Figure 1); ¹H NMR (CDCl₃): δ 2.01 (2H, m, CH₂), 2.28 (1H, t, J=7.2 Hz, H₂-2), 2.21 (2H, m, CH₂), 1.68 (2H, m, CH₂), 1.60 (2H, m, CH₂), 1.51 (2H, m, CH₂), 0.89 (3H, t, J=6.4 Hz, Me-24), 1.30 (8H, brs, 4 x CH₂), 1.28 (2OH, brs, 10 x CH₂), 1.25 (18H, brs, 9 x CH₂), 1.14 (8H, brs, 4 x CH₂), 4.27 (1H, t, J=6.2 Hz, H₂-1ꞌ), 0.86 (3H, t, J=6.8 Hz, Me-13`) (Figure 2); <sup>13</sup>C NMR (CDCl<sub>3</sub>): δ 172.69 (OCO), 64.56 (CH<sub>2</sub>-1ꞌ), 51.40 (CH<sub>2</sub>-2), 38.78 (CH<sub>2</sub>), 34.05 (CH<sub>2</sub>), 28.83 (CH<sub>2</sub>), 33.70 (CH<sub>2</sub>), 31.90 (CH<sub>2</sub>), 30.90 (CH<sub>2</sub>), 29.67 (21 x CH<sub>2</sub>), 29.63 (CH<sub>2</sub>), 29.33 (CH<sub>2</sub>), 29.12 (CH<sub>2</sub>), 10.93 (Me-13`), 26.04 (CH₂), 22.66 (CH₂), 14.06 (Me-24) (Figure 3); LC-MS m/z (rel. int.): 414 [M]⁺ (C₃₇H₇₄O₂) (1.8), 334 (4.8), 350 (4.3) (Figure 4).

Compound 2: Stigmanstan -3β-olyln-octadec-9ʹ, 12ʹ-dienoate

Further when column was eluted with chloroform 100%, it gave a brownish black solid mass (Gowtham et al., 2018) of compound 2 of yield 1.260 mg (0.217% yield) with R_f value 0.31 (Toluene: Ethylacetate (9.3:0.7)); m.p.102-104°C; IR λ max (KBr): 2929, 2862, 1721, 1645, 1459, 1383, 1272, 1209, 1123, 1039, 755 cm^-1 (Figure 5); ¹H NMR (CDCl₃): δ 4.71 (1H, brm, w_1/2 16.8 Hz, H-3α), 5.36 (1H, m, H-10ʹ), 2.03 (2H, m, H₂-8ʹ), 0.71 (3H, brs, Me-18), 5.32 (1H, m, H-12’), 5.17 (1H, m, H-9ʹ), 2.31 (2H, t, J= 7.2 Hz, H₂-2ʹ), 5.04 (1H, m, H-13ʹ),1.56 (2H, m, CH₂-6), 2.35 (1H, m, H₂-11ʹ), 1.94 (2H, m, H₂-14ʹ), 1.89 – 1.60 (25H, m, 9 x CH₂, 7 x CH), 0.80 (3H, t, J= 6.3 Hz, Me-29), 1.42 (1H, m, H-5), 0.89 (3H, d, J= 6.4 Hz, Me-27), 1.33 (2H, m, CH₂), 0.86 (3H, J= 7.2 Hz, Me-18ʹ), 1.30 (6H, brs, 3 x CH₂), 1.28 (6H, brs, 3 x CH₂), 1.25 (8H, brs, 4 x CH₂), 1.18 (3H, brs, Me-19), 0.95 (3H, d, J= 6.2 Hz, Me-21), 0.92 (3H, d, J= 7.2 Hz, Me-26) (Figure 6); ¹³C NMR (CDCl₃): δ 33.11 (C-1), 55.88 (C-14), 68.17 (C-3), 38.73 (C-4), 22.98 (C-6), 34.11 (C-8), 24.94 (C-16ʹ), 30.36 (C-2), 32.05 (C-7), 51.44 (C-9), 35.62 (C-10), 21.03 (C-11), 39.06 (C-12), 42.39 (C-13), 24.19 (C-15), 130.88 (C-12ʹ), 27.24 (C-16), 55.83 (C-17), 10.96 (C-18), 18.70 (C-19), 38.62 (C-20), 17.39 (C-21), 32.97 (C-22), 23.75 (C-23), 45.83 (C-24), 31.92 (C-25), 39.81 (C-5), 19.03 (C-26), 19.82 (C-27), 23.07 (C-28), 11.97 (C-29), 167.77 (C-1ʹ), 25.56 (C-15ʹ), 29.69 (C-2ʹ), 28.93 (C-6ʹ), 22.68 (C-17ʹ), 29.35 (C-4ʹ), 132.45 (C-10ʹ), 29.15 (C-7ʹ), 29.55 (C-8ʹ), 33.96 (C-11ʹ), 29.15 (C-5ʹ), 121.26 (C-13ʹ), 29.61 (C-3ʹ), 29.25 (C-14ʹ), 14.11 (C-18ʹ) (Figure 7); LC-MS m/z (rel. int.): 805 [M]⁺ (C₄₇H₈₂O₂) (1.2), 415 (25.3), 366 (16.6) (Figure 8).

Compound 3: n-Tetracosanol

On continuing the elution with chloroform 100%, it furnished a dark black solid mass of compound 3 of yield 1.820 mg (0.313% yield) with R_f value 0.82 (Toluene: Ethylacetate (9.3:0.7)); m.p.75-77°C; IR λ max (KBr): 3368, 2925, 2854, 1453, 1378, 1219, 1038, 773 cm^-1 (Figure 9); ¹H NMR (CDCl₃): δ 1.25 (38H, brs, 19 x CH₂), 3.42 (2H, t, J=9.5 Hz, H₂-1), 1.54 (4H, m, 2 xCH₂), 0.85 (3H, t, J=6.5Hz, Me-24), 1.87 (2H, m, CH₂) (Figure 10); ¹³C NMR (CDCl₃): δ 64.15 (CH₂-1), 35.68 (CH₂), 33.42 (CH₂), 22.68 (CH₂), 31.26 (CH₂), 29.06 (12 x CH₂), 27.36 (CH2), 28.93 (CH₂), 28.81 (CH₂), 14.16 (Me-24)28.63 (CH₂), 26.51 (CH₂), 25.13 (CH₂) (Figure 11); LC-MS m/z (rel. int.): 354 [M]⁺ (C₂₄H₅₀O) (11.2) (Figure 12).

Results and Discussion

This work resulted in the isolation of three novel compounds from dried roots of Premna latifolia Roxb.

Compound 1: n-Tridecanyl n-Tetracosanoate

Compound 1, a fatty ester, showed bands for ester group (1740 cm^-1) and long aliphatic chain in IR absorption spectra. Its mass spectra exhibited a molecular ion peak at mass/charge 550 which complements to a molecular formula C₃₇H₇₄O_2, a fatty ester. The ion peaks emerging at m/z 351(C₁-O fission, [CH₃ (CH₂)₂₂CO]⁺) and 367 (C₁ʹ-O fission, [CH₃(CH₂)₂₂COO]⁺) designated that lignoceric acid and n-tridecanol were esterified together. The¹HNMR spectra of compound 1 displayed proton triplet at δ 4.27 (J=6.0 Hz) assigned to oxygenated methylene H₂-1ʹ proton. A two proton triplet in the upfield region at δ 2.28 (J= 7.2 Hz) was associated to methylene H₂-2 proton. Other protons of methylene resonated between δ 2.20- 1.14. The proton triplets at δ 0.89 (J= 6.4 Hz) and 0.86 (J= 6.8 Hz) were accounted to terminal C-24 and C-13ʹ primary methyl protons. ¹³CNMR spectra of compound 1 showed ester carbon signals at δ 172.61 (C-1), oxygenated methylene carbon at δ 64.56 (C-1ʹ), other methylene carbons from δ 51.40 to 22.66 and methyl carbons at δ 14.0 (C-24) and 10.93 (C-13ʹ). The ¹H-¹H COSY spectra exhibited correlations of H₂- 22 and H₂- 23 with Me- 24; H₂-1ʹ with H₂-2ʹ and H₂-3ʹ; and H₂- 12ʹ with Me-13ʹ. The HMBC spectra of compound 1 indicated interaction of H₂-2ʹ and H₂-3ʹ with C-1ʹ; H₂-23 with C-24; H₂-2 and H₂-23 with C-1 and H₂-12ʹ with C-1ʹ. Based on the above data, the name of Compound 1 is suggested as-n-Tridecanyl n-Tetracosanoate (Figure 13).

Compound 2: Stigmanstan -3β-olyln-octadec-9ʹ, 12ʹ-dienoate

IR spectra displayed characteristic bands attributed for unsaturation (1645 cm^-1), ester group (1721 cm^-1) and aliphatic chain, which is long (755 cm^-1). From mass spectra, the molecular ion peak was determined at m/z 678, and ¹³C NMR spectra were correlated with the molecular formula of a sterol ester C₄₇H₈₂O₂. The peaks arising at m/z 415 [CO-O fission, C₂₉H₅₁O]⁺ and 279 [M]⁺- 415, CH₃(CH₂)₇CH=CHCH₂-CH=CH(CH₂)₄COOH]⁺ accredited that sterol was esterified with the linoleic acid. The ¹H NMR spectra of compound 2 displayed four one-proton multiplets from δ 5.36 to 5.04 adduced to vinylic H-9ʹ, H-10ʹ, H-12ʹ and H-13ʹ protons. A multiplet one-proton broad at δ 4.71 with a half-width of 16.8 Hz was associated to oxygenated methine H-3 proton (α-oriented). A 2 proton multiplet and triplet at δ 2.35 and δ 2.31 (J=7.2 Hz) respectively, were due to methylene H₂-11ʹ between two vinylic carbons and to methylene H₂-2ʹ protons which were adjacent to the ester function. An 3- proton doublets at δ 0.95 (J=6.2 Hz), 3-proton triplets at δ 0.86 (J= 7.2 Hz) and 0.80 (J= 6.3 Hz) and 3 proton broad singlets at δ 1.18 and 0.71, 0.92 (J=7.2 Hz), 0.89 (J=6.4 Hz) were assigned with tertiary C-19 and C-18 secondary C-21, C-26 and C-27 and primary C-18ʹ and C-29 methyl protons, respectively, all attached to the saturated carbons. The remaining methylene and methine protons resonated from δ 2.03- 1.25. The ¹³C NMR spectra of compound 2 displayed presence for vinylic carbons at δ 126.90 (C-9ʹ), 132.45 (C-10ʹ), 130.88 (C-12ʹ) and 121.26 (C-13ʹ), oxygenated methylene carbon at δ 68.17 (C-3), ester carbon at δ 167.77 (C-1ʹ) and methyl carbons between δ 18.70 –10.96. The ¹H-¹H COSY spectra of compound 2 showed correlations of H-3 with H₂-1, H₂-2, H₂-4 and H-5; H-8 with H₂-6, H₂-7, H-9 and H_-14; H-17 with H₂-16, H₂-22, H-20 and Me-21; H-24 with H₂-23, H₂-28, H-25, Me-26, Me-27 and Me-29; and H₂-11 with H-9ʹ, H-10ʹ, H-12ʹ and H-13ʹ. The HMBC spectra of compound 2 exhibited associations of H-3 and H₂-2ʹ with C-1ʹ; H₂-1, H₂-2, H₂-4 and H-5 with C-3; H₂-1, H-5, H-9 with C-19; H-8 with H₂-6, H₂-7, H-9 and H_-14; H₂-12, H-14, and H-17 with C-18; H-17, H₂-22 and Me-21 with C-20; and H₂-23, H₂-28, H-25, Me-26, Me-27 and Me-29 with C-24. The ¹³C NMR and ¹H spectral data of the steroidal nucleus obtained were compared with all the reported spectral steroidal values. Based on these information, the structure of compound 2 was elucidated and confirmed as stigmanstan -3β-olyl n-octadec-9ʹ, 12ʹ-dienoate (Figure 14), (Figure 15). This is a new sterol ester.

Compound 3: n-Tetracosanol

The IR spectra of compound 3 showed at 3368 cm^-1, which suggest the presence of OH group along with a long aliphatic chain at 773 cm^-1. The mol. ion peak at m/z 354 [M]⁺ in mass spectra exhibited a molecular formula C₂₄H₅₀O. The ¹H NMR spectra showed the presence of a two proton triplet at δ 3.42(J= 9.5 Hz) and a 3-proton triplet at 0.85 (J= 6.5 Hz) adduced to hydroxy methylene H₂-1 and terminal C-24 primary methyl protons respectively. The methylene protons were found to resonate as multiplets at δ 1.87 (2H) and δ 1.54 (4H) and as abroad singlet at δ 1.25 (38H). The ¹³C NMR spectra of compound 3 displayed signals for hydroxy methylene carbon at δ 64.15 (C-1), methylene carbons between δ 35.68- δ 22.68 and methyl carbon at δ 14.16 (C-24). The ¹H - ¹H COSY spectra showed correlations of H₂- 1 with H₂- 2 and H₂-3 and Me-24 with H₂ – 23 and H₂ -22 respectively. The HMBC spectra of compound 3 exhibited interactions of H₂- 2 and H₂-3 with C-24. Based on the above datas and discussion, the structure of compound 3 has been confirmed as n-Tetracosanol (Figure 16).

Conclusion

Plants are blessed with the ability to produce a large number of diverse bioactive and interesting compounds with important pharmacological activities. The present work led to isolation and characterization three compounds, namely Compound 1: n-Tridecanyl n-Tetracosanoate, Compound 2: Stigmanstan -3β-olyln-octadec-9ʹ, 12ʹ-dienoate and Compound 3: n-Tetracosanol which possess the significant pharmacological activity and can be drugs of future.

Acknowledgement

The authors express their sincere thanks to Hon. Chancellor, VIT, Vellore, Prof. Mary Saral, Dean SAS, VIT, Vellore; Management, Rajiv Gandhi Institute of Pharmacy, Trikaripur-671310, Kasaragod (Dist.), Kerala; SAIF, Panjab University, Dr. Mohd. Ali and Dr. Tanvir Ali for giving all inspiration and valuable support to carry out this research work.