Biological Importance of Phytoconstitents Isolated from the Genus Randia & GC-MS Analysis of Petroleum-Ether Fruit Extract of Randia dumetorum

Randia genus (Indigo berry) belongs to family Rubiaceae, native to America, allocated in tropical regions. Most of the Species associated with this Genus used as ornamental, folk medicine to treat the disease of renal, malaria, cancer, dysentery, snake-bite etc. All plant parts are used by tribes for various ailment treatments. The phytochemicals generally associated with this genus are terpenoids, saponins, lignans, coumarin, iso-coumarin, flavonoids, tannins, essential oil and acid resin. This review highlights the phytochemicals and pharmacological activity reports. Phytochemical investigation of Randia dumetorum fruit extract using petroleum ether solvent, reveals the presence of 2,4-di-tert-butylphenol, octadecadienoic acid, 23(Phenylsulfanyl) lanosta-8,24-dien-3-ol, hexadecenoic acid, gamma-sitosterol, 4-tert-butylcalix[4]arene, tetracontane, tetratetracontane and octacosanol etc. Compound 1-Octacosanol (A), 9-Octadecenoic acid (B) and gamma-Sitosterol (C) were isolated with the help of column chromatographic techniques and characterized by spectral studies i.e. 1 H NMR, 13 C NMR


INTROdUCTION
Randia genus commonly known as Indigo berry, belongs to Rubiaceae family, sub-family Ixoroideae, tribe-Gardeniae, native to the america, mainly distributed in tropical and subtropical regions.This is a large and well-defined family, with more than 600 genera and about 13,500 species found worldwide [1][2][3] .This genus associated with more than 100 species.Out of them more than 10 species are ethnopharmacologically important.Randia echinocarpa, Randia matude, Randia aculeata, Randia hebecarpa, Randia ferox, Randia momantha are the most studied species 4 .Excessive ethnobotanical and ethnomedicinal study of root, bark, leaves, fruit and seeds of this genus are reported in literature.
Only Randia aculeata shows the antivenom effect 4 .Pseudogenosides and tyramine isolated from Randia siamensis are responsible for hypotensive and hypertensive activity respectively.Fruits of Randia echinocarpa (native to the maxico) consumed by rural areas as food/medicine 4 .

Collection & pre-treatment of fruit sample
The fruit samples of Randia dumetorum fruit were collected from local market of Jaipur city Rajasthan (India).The fruit sample were washed, cleaned and dried.The plant's identification was confirmed by Dr. Mahesh C. Sharma (Retd.Professor), Department of Chemistry, University of Rajasthan, Jaipur.

Preparation of extracts and Column Chromatography
The air dried Randia dumetorum fruits were ground to fine powder using a grinder.Then the powdered fruits (approximately 1 kg) of Randia dumetorum were subjected to Soxhlet extraction procedure using Petroleum ether solvent (3ltr), for 12×3 days."Rotary Vacuum Evaporator (N.N. Series) with a digital water bath SB-651 and an aspirator; Eyela, tokyo, Japan'' was used to remove excess solvent and further sodium sulphate remove moisture from extract.Above extract was stored in air tight container at low temperature.It was filtered by Whatman No.1 filter paper to remove grainy matter and concentrated sample of extract was obtained.The extract was dissolved in petroleum ether (20-30 mg/mL) for GC-MS analysis.Fat free extract (after acetonitrile treatment) was subjected to column chromatographic separation over a silica gel column.Column elution was done by different solvent system according to increasing polarity.

GC-MS analysis
GC-MS analysis of Randia dumetorum fruit extract (Petroleum ether) was done at the Advanced Instrumentation Research Facility (AIRF) Lab, JNU, New Delhi using standard GCMS model as explained below.

Instrument details
Shimadzu GCMS-QP2010 Plus with thermal desorption system TD 20, AOC-20S auto-sampler & 20i auto-injector, was used to performed Gas chromatography-mass spectroscopy (mass range 1.5-1090 Daltons).The total run time of GC-MS was 29 minutes.Different compounds were detected by mass spectrometer according to their different retention time.A plot of intensity v/s retention time was recorded (Chromatogram).The compounds are identified by comparing the data with the WILEY8, NIST14 & NIST14s libraries with name, molecular formula, molecular weight and structures.

Isolation of compound C as g-sitosterol
On eluting the column with chloroform white needle like crystals obtained.It showed
The 1 H NMR spectrum (dppm, CDCl 3 ), exhibited abroad triplet at 5.32 corresponding to H-6 olefinic proton.A multiplet appeared at 3.50 for H-3 a-proton.The rest of the protons of compound appeared in high field region 0.66-2.42.
In 13

Characterization of compound B as 9-Octadecenoic acid
Cream-coloured oil obtained when column was eluted with pet-ether and benzene in ratio 3:1.m.p. 10-12 0 C. Its IR spectrum (KBr, cm -1 ) appeared at 2950 confirmed the presence of carboxylic group.The absorption at 1712 due to carbony group whereas the absorption at 1620 confirmed the presence of olefinic (C=C stretching) group.
The 1 H NMR spectrums (dppm, CDCl 3 ) showed a sharp singlet for one proton of carboxylic group at 10.48 (s, 1H, -COOH).A triplet at 2.23 (t, 2H, C-2) was assigned for C-2 protons and a multiplet was observed at 1.58-1.62(m, 2H, C-3) due to protons of C-3 position.A broad singlet at 1.28-1.30integrated for 20 protons of ten methylene group at C-4 to C-7 and C-12 to C-17 respectively.A multiplet observed at 1.89-1.95was assigned to the protons of methylene present at C-8 and C-11 positions.Protons attached to olefinic carbon at position C-9 and C-10 showed a multiplet at 5.24.A triplet at 0.93 (t, 3H, C-18) for three protons of methyl group at C-18 position.From the above spectral data, it was identified as compound "2" 9-Octadecenoic acid.

9-Octadecenoic acid Characterization of Compound C as gamma-Sitosterol
The compound 3 was isolated as white powder, gave positive Liebermann-Burchard and Salkowski tests characterstic for sterols.The mass

CONCLUSION
Defatted petroleum-ether extract produced 1-octacosanol (A), Octadecanoic acid (B) andν g-sitosterol (C).In view of the immense biological, nutraceutical and pharmacological importance of the genus randia, we have systematically reviewed this genus as it may be helpful to food industry and