Daruharidra (in Sanskrit means ‘the wood having yellow color’) is one of the herbs mentioned in ancient scriptures of Ayurveda. Charaka has categorized Daruharidra as stanyasodhana (lactone purant), lekhana (a reducing herb), arsoghna (antihaemorrhoidal), kandughna (antipruritic), svedala (promotes sweating) and rasayana (rejuvenative). Susruta has mentioned it as ropana–a wound healer. Ayurvedic Pharmacopoeia of India correlates Daruharidra to Berberis aristata DC var. aristata (Berberidaceae), which is a spinous shrub native to northern Himalaya region. The plant is widely distributed from Himalayas to Srilanka, Bhutan and hilly areas of Nepal. It grows at the height of 2000-3000 m especially in Kumaon and Chammba region of Himachal Pradesh [1]. Phytochemical studies have shown that plant B. aristata contains berberine- the chief alkaloidal constituent, oxyberberine, berbamine, aromoline, a protoberberine alkaloid karachine, palmatine, oxycanthine, taxilamine, tannins, sugar and starch. E-caffeic acid, quercetin, chlorogenic acid, meratin, rutin have been isolated from the flowers of B. aristata [2]. Its various parts have been found to be beneficial as anti-inflammatory [3,4], anti-microbial [5-7], anti- tumor [8-10], anti-diabetic [11,12], antidiarrhoeal [13], anti-hepatotoxic [14], anti-pyretic [15], anti-oxidant [16] and anti-osteoporotic agent [17]. It has also shown to be effective in ear infections [1] and skin ageing [18].

The melting point of the compound was determined on Centigrade scale in one-end open capillary using Perfit melting point apparatus. IR spectra were recorded on Perkin Elmer spectrum RX 1 model. 1H and 13C-NMR spectra were scanned on Bruker DRX-300 NMR (300 MHz) instrument in CDCl₃ and D₂O using Tetramethylsilane (TMS) and CDCl₃ as the internal standard. The coupling constants (J values) are expressed in hertz (Hz). Mass spectra were recorded by affecting electron impact ionization at 70 eV on a Jeol SX-102 (FAB) mass spectrometer equipped with direct inlet probe system. The m/z values of the more intense peaks are mentioned and the figures in brackets attached to each m/z values indicate relative intensities with respect to the base peak. The solvents used were of Qualigens LR grade. Silica gel (Qualigens 60-120 μm mesh) was used for column chromatography.

TLC was performed on plates coated with silica gel G (Qualigens). Anhydrous sodium sulphate was used for drying all the solvents used during the research work.

The plant material was procured from AIMIL Pharmaceuticals, New Delhi. It was authenticated as Berberis aristata by Dr. M.P. Sharma, Reader, Department of Botany, Jamia Hamdard, New Delhi and a voucher specimen is preserved in the herbarium section of Department of Pharmacognosy, Ram-Eesh Institute of Technology, Greater Noida, and Uttar Pradesh. The plant material (1.8 kg) was air dried crushed to smaller pieces, coarsely powdered and was then exhaustively extracted with ethanol (95%) in a Soxhlet Apparatus for 72 hr. The ethanolic extract was dried and dark brown mass, 50 gm (2.77%) was obtained. The concentrated extract of the drug was taken in a china dish and heated continuously on a water bath, gradually adding methanol in small portions with constant stirring till desired consistency was obtained. Weighed quantity of silica gel (60-120 mesh) was added slowly by mixing with a stainless steel spatula to obtain the desired consistency. It was dried in air; the larger lumps were brokenup and finally passed through a sieve (No.8) to get a uniform particle size. The lower end of a clean dry column was plugged with adsorbent cotton. The column was then half filled with petroleum ether. Silica gel was added in small proportions and allowed to settle down gently until the necessary length of the column was attained. All the air bubbles were allowed to escape by running the column blank thrice with solvent. The dried silica gel slurry of the extract was packed in the column and plugged with the adsorbent cotton and then eluted successively in the order of increasing polarity with different solvents. The development and elution of the column was carried out with successive series of solvents in various combinations, viz., petroleum ether, chloroform in petroleum ether (0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%) chloroform (100%), and methanol in chloroform. The fractions collected were subjected to Thin Layer Chromatography. Chromatographically identical fractions were combined and concentrated. Elution of the column with chloroform gave colourless crystals of a compound recrystallized from methanol: chloroform (1:1), yield 85 mg (0.17%).

Rf: 0.49 (benzene: chloroform, 1:1); m.p.: 600°C - 610°C; IR υmax (KBr): 2963, 2930, 2869, 1725, 1629, 1493, 1277, 1215, 1080, 970, 909, 756cm^-1 ; ¹HNMR(CDCl₃): δ 7.55 (2H, m, H^-2’, H^-6’), 7.36 (2H, m, H^-3’, H^-5’), 7.15 (¹H, m, H^-4’), 4.27 (¹H, d, J=11.6 Hz, H2 – 1a), 4.23 (¹H, d, J=11.6 Hz, H2 – 1b), 1.57 (2H, m, CH₂), 1.33 (14H, brs, 7 × CH₂), 6 1.29 (20H, brs, 10 × CH₂), 1.23 (8H, brs, 4 × CH₂), 1.12 (2H, brs, CH₂), 0.88 (3H, t, J=6.3 Hz, Me-24); +ve ion FAB MS m/z (rel. int.): 458[M]+ (C³¹H⁵⁴O²)(73.1), 443(14.2), 387(8.9), 345(12.6), 331(12.9), 317(11.2), 303(13.5), 289(13.7), 219(22.3), 205(18.6), 191(91.4), 135(20.3), 121(26.2), 105(71.6).

The Compound (Figure 1) was obtained as a colourless crystalline mass from chloroform eluents. Its IR spectrum (Figure 2) showed characteristic absorption bands for ester group at 1725 cm^-1, aromatic ring at 1629, 970 cm-1 and a long aliphatic chain at 756 cm^-1. The ¹HNMR spectrum (Figure 3) exhibited signals for aromatic protons. The mass spectrum (Figure 4) displayed a molecular ion peak at m/z 458 corresponding to the benzyl ester, C₃₁H₅₄O₂. The ion fragments (Figure 5) arising at m/z 105(C₆H₅CO)+ and 121(C₆H₅COO)+ indicated that benzoyl function was present in the molecule. The ion peaks generating at m/z 135(C₆H₅COOCH₂)+, 205(C₆H₅COO(CH₂)₆)+, 219(C₆H₅COO (CH₂)7)+,289(C₆H₅COO(CH₂)12)+, 303(C₆H₅COO(CH₂)₁₃)+, 317(C₆ H₅COO(CH₂)₁₄)+, 331(C₆H₅COO(CH₂)₁₅)+, 345(C₆H₅COO(CH₂)₁₆)+, 387(C₆H₅COO(CH₂)₁₉)+ and 443(C₆H₅COO(CH₂)₂₃)+ supported the presence of the aromatic ring at one of the terminal carbon of the aliphatic chain. The 1HNMR spectrum (Figure 3) exhibited signals for aromatic protons as multiplets at δ 7.55 (H-2’, H-4’). Two oneproton doublets at δ 4.27 (J=11.6Hz) and 4.23 (J=11.6Hz) were ascribed to oxygenated methylene protons H2- 1’. A three-proton triplet at δ 0.88 (J=6.3Hz) was accounted to C-24 primary methyl protons. The remaining methyl protons resonate between δ 1.57-1.12. Alkaline hydrolysis of the compound yielded benzoic acid. On the basis of foregoing discussion the structure of the compound has been formulated as n-tetracosanyl benzoate.

Figure 1: Structure of n-Tetracosanyl Benzoate.

Figure 2: IR spectra of compound n-Tetracosanyl Benzoate.

Figure 3: ¹H NMR spectra of compound n-Tetracosanyl Benzoate.

Figure 4: Mass spectra of compound n-Tetracosanyl Benzoate.-

Figure 5: Mass Fragmentation Pattern of n-tetracosanyl benzoate.

A novel phytoconstituent “n-tetracosanyl benzoate” was isolated and characterized from ethanolic extract of the heartwood of Berberis aristata. The chemical structure was elucidated by means of various physical (solvent extraction, TLC, Column chromatography) and spectral techniques. B. aristata has been found to be beneficial as antiinflammatory, anti-microbial, anti-tumor, anti-diabetic, anti-diarrheal, anti- hepatotoxic, anti-pyretic, anti-oxidant, anti-osteoporotic agent etc. due its diverse phyto constituents. So, n-tetracosanyl benzoate might also be an active constituent possessing some pharmacological activity. This compound can be useful object for some future research work.