Chemical Constituents of Cordia dichotoma G . Forst .

Consolacion Y. Ragasa, Virgilio Ebajo Jr., Mariquit M. De Los Reyes, Emelina H. Mandia, Maria Carmen S. Tan, Robert Brkljača, Sylvia Urban Chemistry Department, De La Salle University Science & Technology Complex Leandro V. Locsin Campus, Biñan City, Laguna 4024, Philippines. Chemistry Department, De La Salle University, 2401 Taft Avenue, Manila 1004, Philippines. Biology Department, De La Salle University Science & Technology Complex Leandro V. Locsin Campus, Biñan City, Laguna 4024, Philippines. Biology Department, De La Salle University, 2401 Taft Avenue, Manila 1004, Philippines. School of Applied Sciences (Discipline of Chemistry), RMIT University (City Campus), Melbourne 3001, Victoria, Australia.


INTRODUCTION
Cordia dichotoma G. Forst.belongs to the family Boraginaceae.This medium sized tree grows widespread in the Philippines where it is locally known as anonang.It yields edible fruits from which a very sticky white substance could be extracted for gluing purposes.The leaves, fruit, bark and seed have been reported to exhibit antidiabetic, antiulcer, antiinflammatory, immune-modulator and analgesic activities (Jamkhande et al., 2013).The leaves, seed, fruit, bark, and roots are well-known to have different medicinal uses: antidiabetic, anthelmintic, antilarvicidal, hepatoprotective, immunomodulator, antidysentery, antidyspepsia, anti-inflammatory, antiulcer, diuretic and laxative (Patel et al., 2011).The crude ethanol extract from the leaves showed antilarvicidal potency against the brine shrimp Artemia salina (Sharker et al., 2013).Initial studies indicated potential antioxidant properties based on direct measurement of radical scavenging activity of crude extracts from the tree's bark (Nariya et al., 2013).Crude methanolic extract of the bark showed inhibition of number of implants in laboratory female rats (Katolkar et al., 2012).In a related study, leaf extract from the tree showed anti-implantation activity, suggesting its possible use as a natural contraceptive drug (Bhattacharya and Saha, 2013).
The fruit, leaves and seed contain pyrrolizidine alkaloids, coumarins, flavonoids, saponins, terpenes and sterols (Jamkhande et al., 2013).Taxifolin from the seeds of C. dichotoma showed promising DPPH free radical scavenging activity at a concentration of 100 μg/mL (Mahasweta et al., 2014).Another study reported that apigenin (5 mg/kg, p.o.) from C. dichotoma showed significant healing and reduction in inflammatory enzymes when screened for ulcerative colitis (Ganjare et al., 2011).The fatty oil content in the seeds of C. dichotoma was found to be 7.60% with stearic, oleic and linoleic acids as the major constituents (Rameshwar et al., 2006).Three flavonoids, kaempferol, quercetin and isorhamnetin, were isolated from the butanol fraction of fruits of C. dichotoma (Kuppast et al., 2006).
Six flavonol glycosides and two phenolic compounds were isolated from butanol extract of the leaves of C. dichotoma with rosmarinic acid as a major constituent (Wang et al., 1996).In another study, the ethanolic extracts were reported to contain polyphenolic compounds (1.0%), triterpenoids (0.075%), amino acids (1.39%), and rosmarinic acid (0.0028%) (Tian et al., 2014).A review on the chemical constituents and medicinal uses of .

General Experimental Procedure
1 H (500 MHz) and 13 C (125 MHz) NMR spectra were acquired in CDCl 3 on a 500 MHz Agilent DD2 NMR spectrometer with referencing to solvent signals (δ 7.26 and 77.0 ppm).Twodimensional NMR experiments recorded included gCOSY, HSQCAD, and gHMBCAD NMR experiments.Column chromatography was performed with silica gel 60 (70-230 mesh).Mass spectral analysis was conducted on the Bruker Daltonics micro TOF Q -II mass spectrometer employing ESI -Qq -TOF in positive mode.Data analysis was processed using the Bruker Compass Data Analysis 4.0 application.
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 leaves and twigs of Cordia dichotoma G. Forst.were collected from the De La Salle University -Science and Technology Complex (DLSU-STC) riparian forest in February 2014.The samples were authenticated by one of the authors (EHM) and deposited at the De La Salle University Herbarium with voucher specimen # 916.

General Isolation Procedure
The air-dried leaves (300 g) and stems (121.4 g) of C. dichotoma were ground in a blender, soaked in CH 2 Cl 2 for three days and then filtered.The filtrates were concentrated under vacuum to afford crude extracts of leaves (6.3 g), and stems (1.0 g) which were each chromatographed by gradient elution with CH 2 Cl 2 , followed by increasing amounts of acetone at 10% increment by volume as eluents.A glass column 12 inches in height and 0.5 inch internal diameter was used for the fractionation of crude extracts.
Two milliliter fractions were collected.Fractions with spots of the same R f values were combined and rechromatographed in appropriate solvent systems until TLC pure isolates were obtained.Subsequent chromatography and final purifications were conducted using Pasteur pipettes as columns.One milliliter fractions were collected.

RESULTS AND DISCUSSION
Silica gel chromatography of the dichloromethane extracts of C. dichotoma yielded 1-4 from the leaves; and 3 and 5 from the twigs.The structure of 1 was identified by comparison of its 1 H and 13 C NMR data (Table 1) with those reported in the literature for β-sitosteryl-3β-glucopyranoside-6'-O-palmitate (1) (Nguyen et al., 2004).
β-Sitosterol (3) was observed to have growth inhibitory effects on human breast MCF-7 and MDA-MB-231adenocarcinoma cells (Awad et al., 2007).It was shown to be effective for the treatment of benign prostatic hyperplasia (Jayaprakasha et al., 2007).It was also reported to attenuate βcatenin and PCNA expression, as well as quench radical in-vitro, making it a potential anticancer drug for colon carcinogenesis (Baskar et al., 2010).It can inhibit the expression of NPC1L1 in the enterocytes to reduce intestinal cholesterol uptake (Jesch et al., 2009).It was reported to induce apoptosis mediated by the activation of ERK and the downregulation of Akt in MCA-102 murine fibrosarcoma cells (Moon et al., 2007).

CONCLUSION
Cordia dichotoma is a medicinal tree with many reported biological activities.This study reports on the isolation of compounds (1-5) from the dichloromethane extracts of the leaves and stems of C. dichotoma.Compounds 1-4 have been reported to possess anticancer and cytotoxic properties.The antioxidant property of 2, cholesterol lowering effect of 3 and wound healing effect of 4 have also been reported.Thus, the medicinal properties of C. dichotoma maybe partly attributed to 1-4 which were reported to exhibit diverse biological activities.