Chromatographic Isolation and Structure Elucidation of New Phenolic Glycosides from Ocimum Sanctum L. Leaves

Ocimum sanctum L. (Lamiaceae) leaves are used to treat chronic fever, dysentery, hemorrhage, dyspepsia, vomiting, malaria, skin diseases, muscle pain and respiratory infections in Indian traditional system of medicine. Four new aromatic phenolic glycosides were isolated from the methanolic extract of leaves of O. sanctum and their structure were elucidated as 4-(4′octadec-9′′,12′′,15′′-trienoyl-β-O-D-glucopyranosyl) ferulic acid (1), 3,3′-dimethoxy-4,4′-dilinolenyl rosmerinic acid (2), 3-methoxy-4-α-O-D-arabinopyranosyl-5-decanoyl gallic acid (3) and 3-methoxy-4-β-O-D-arabinopyranosyl-(2′→1′′)-β-O-Dglucopyranosyl-(2′′→1′′′)-β-O-D-glucopyranosyl-5-octadecanosyl gallic acid (4). The methanolic extract of O. sanctum leaves was obtained by Soxhlet extractor. Isolation of compounds was done by silica gel column chromatography and analytical thin layer chromatography. The structure of new compounds was established on the basis of chemical reactions and extensive spectroscopic analysis (NMR, FT-IR, UV and MS).


Introduction
Ocimum sanctum L. (Lamiaceae) is a sacred medicinal plant with specific aroma commonly known as tulsi or sacred basil. The genus Ocimum includes about 50-150 species with a number of varieties different in their chemical profile but characterization of them is based on leaves and habitat [1][2][3][4]. It is an annual, erect and much branched herb and its leaves are elliptic oblong, obtuse or acute, entire or serrate, minutely gland dotted, pubescent on the both sides with obtuse or acute base. It is a popular herbal medicine and distributed all over the India ascending up to 1,800 m in Himalayas and found down in the tropic areas, Andaman and Nicobar Islands, Pakistan, Nepal, western Asia, Saudi Arabia, Malaya, Australia, Phillipines, Brazil, Iran and Egypt [5][6][7]. O. sanctum is considered as a kind of 'elixir of life' and believed to promote longevity. It has been used for thousands of years for its various medicinal properties as it balances different processes in the human body and helpful for adapting to stress [8]. O. sanctum dried leaves and extracts are available commercially in the form of powder, tablets and capsules alone or in combination with other medicinal herbs (polyherbal formulations). Traditionally, O. sanctum leaves are used to treat chronic fever, cough and cold, malaria, skin diseases, gastric and hepatic disorders and recommended as anthelmintic, diaphoretic, antiperiodic, expectorant, antiemetic and antispasmodic [1,5,9]. The major constituent of the leaves is a volatile oil mainly consisted of phenylpropanoids, eugenol and methyl eugenol [10,11]. Previous phytochemical studies reported the presence of triglycerides, terpenoids, hydroxyl cinnamic and benzoic acid derivatives, fatty acids, flavonoids and their glycosides, phenyl propane glycosides, glycoglycerolipids and coumarins in the plant. Some of these compounds possess antimicrobial,antioxidant,antistress,antidiabetic,anthelmintic,radioprotective,anticancer,. In continuation of our previous work [18], this article reports the chromatographic isolation and characterization of new phenolic glycosides from the O. sanctum leaves collected from Delhi region of India.

General
Melting points were determined on a Perfit apparatus (India) 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 UV-Vis spectrophotometer (Shimadzu, Japan). 1 H (300 MHz) and 13 C (75 MHz) nuclear magnetic resonance (NMR) spectra were recorded on Bruker300 spectrospin spectrometer (Karlsruhe, Germany). CDCl 3 and DMSO-d 6 (Sigma-Aldrich, Bengaluru, India) were used as solvents and TMS as an internal standard. ES MS analyses were performed on a Micromass Quattro II triple quadrupole mass spectrometer. Column chromatography separations were carried out on silica gel (Merck,Mumbai,India). Precoated silica gel plates (Merck, Silica gel 60 F 254 ) were used for analytical thin layer chromatography and visualized by exposure to iodine vapours and UV radiations.

Plant Material
The leaves of O. sanctum were collected from the herbal garden of Jamia

Extraction and Isolation of Compounds
The air dried coarsely powdered leaves (500 gm) were extracted exhaustively with methanol (2 L) through continuous hot extraction process for 24 h. The extract was filtered through Whatman qualitative filter paper and concentrated under vacuum (337 mbar) at 40°C to yield a brown mass (183 g). The extract was redissolved in methanol and treated with equal volume of aqueous lead acetate (10%) to precipitate tannins and other impurities and filtered. The filtrate was defatted with petroleum ether (500 ml x 3) and re-dried. The residue (80 g) was dissolved in minimum amount of methanol and adsorbed on silica gel column grade (60-120 mesh) to obtain slurry. The slurry was dried in air and chromatographed over silica gel column loaded in chloroform [19]. The column was eluted with chloroform-methanol (99:1, 17:3 and 4:1) mixtures to obtain compounds 1-4 ( Figure 1). Analytical TLC was used to check the homogeneity of eluted fractions.

Ocimumglucoferulic Acid (1)
Elution of the column with chloroform-methanol

Conclusion
The systematic chemoprofiling of medicinal herbs with the purpose of discovering new natural compounds is a routine research activity. The research on the medicinal herbs should be extended with the isolation, identification and structure elucidation of natural compounds. The present work reports the chromatographic isolation and spectroscopic characterization of four new phenolic glycosides identified as ocimumglucoferulic acid, dilinolenyl rosmerinic acid, arabinogallic acid ester and triglycogallic acid ester from the methanolic extract of O. Sanctum dried leaves. The isolated compounds have enhanced the phytochemical nature of plant and may be used as chromatographic markers for quality control assessments of crude plant material and its commercial herbal formulations. The medicinal properties of O. sanctum have been reported in number of scientific studies including in vitro, preclinical and clinical experiments. These findings revealed that it has a unique combination of actions due to its natural compounds. Keeping in view further studies are needed to determine the biological potential of these phenolic compounds in future.