Chemical Constituents of Cymodocea rotundata Asch. and Schweinf

Introduction: Cymodocea rotundata Asch. and Schweinf, a widespread seagrass with reported antimicrobial activity, was investigated for its chemical constituents. Methods: The compounds were isolated by silica gel chromatography and identified by NMR spectroscopy. Results: This study has led to the isolation of β-sitosteryl-3β-glucopyranoside-6′-O-fatty acid esters (1), chlorophyll a (2) and a mixture of β-sitosterol (3a) and stigmasterol (3b) in about 1:1 ratio from the dichloromethane extract of C. rotundata. Conclusion: This is the first report on the isolation of 1-3b from C. rotundata. Compounds 2-3b were reported to exhibit antibacterial activity and may be partly responsible for the reported antimicrobial activity of the C. rotundata extract.


INTRODUCTION
Cymodocea rotundata is a widespread seagrass that occurs in shallow water, on sand-mud in sheltered coves or bays, lagoons, mouth of rivers and coral reefs throughout the Philippines. 1 It has a wide Indo-Pacific distribution and is relatively common within its range. 2C. rotundata extracts exhibited predominant growth inhibitory activity against UTI bacteria 3 and human pathogens. 4,5Phytochemical analysis of C. rotundata extracts indicated the presence of tannins, saponins, resins, proteins, acidic compounds, reducing sugars, terpenoids, cardiac glycosides and alkaloids. 4nother study reported that C. rotundata contained vitamin C (28.43 mg g − ¹) and p-coumaric acid.C. rotundata extracts gave a total antioxidant activity of 6.65 mg ascorbic acid equivalent/g and was found to be a potent DPPH radical scavenger (70.30%). 6his study is part of our research on the chemical constituents of seagrasses found in the Philippines.We earlier reported the isolation of bis(2-ethylhexyl) phthalate, chlorophyll a, β-sitosterol and stigmasterol from Cymodocea serrulata. 7We report herein the isolation of β-sitosteryl-3β-glucopyranoside-6'-Ofatty acid esters (1), chlorophyll a (2) and a mixture of β-sitosterol (3a) and stigmasterol (3b) from C. rotundata.To the best of our knowledge, this is the first report on the isolation of 1-3b from C. rotundata.
Thin layer chromatography was performed with plastic backed plates coated with silica gel F 254 and the plates were visualized by spraying with vanillin/ H 2 SO 4 solution followed by warming.

Sample Collection
Samples of the leaves of Cymodocea rotundata Asch.and Schweinf.were collected from the seagrass meadow of Caramoan, Camarines Sur Philippines in September 2016.The samples were authenticated at the Botany Division, Philippine National Museum.

General Isolation Procedure
A glass column 6 inches in height and 0.25 inch internal diameter was used for the chromatography.The crude extracts were fractionated by silica gel chromatography using increasing proportions of acetone in CH 2 Cl 2 at 10% increment by volume as eluents.Five milliliter fractions were collected.All fractions were monitored by thin layer chromatography.Fractions with spots of the same Rf values were combined and rechromatographed in appropriate solvent systems until TLC pure isolates were obtained.Final purifications were conducted using Pasteur pipettes as columns.One milliliter fractions were collected.

Isolation of the Chemical Constituents from the Leaves of C. rotundata
The air-dried C. rotundata (50 g) leaves were ground in a blender, soaked in CH 2 Cl 2 for 3 days and then filtered.The solvent was evaporated under vacuum to afford a crude extract (0.3 g) which was chromato-graphed using increasing proportions of acetone in CH 2 Cl 2 at 10% increment by volume.The 10% acetone in CH 2 Cl 2 fraction was rechromatographed using 10% EtOAc in petroleum ether.The less polar fractions were combined and rechromatographed using 10% EtOAc in petroleum ether to afford 2 (4 mg) after washing with petroleum ether, followed by Et 2 O.The more polar fractions were combined and rechromatographed (2 ×) using 15% EtOAc in petroleum ether to yield a mixture of 3a and 3b (6 mg) after washing with petroleum ether.The 60% acetone in CH 2 Cl 2 fraction was rechromatographed using CH 3 CN:Et 2 O:CH 2 Cl 2 (1:1:8, v/v) to afford 1 (3 mg) after washing with petroleum ether.

CONCLUSION
This is the first report on the isolation of 1-3b from C. rotundata.Compounds 2-3b were reported to exhibit antibacterial activity and may be partly responsible for the reported antimicrobial property of the C. rotundata extract.

ACKNOWLEDGEMENT
A research grant from the De La Salle University Science Foundation through the URCO is gratefully acknowledged.