Bioassay Guided Phyto-chemical Investigation of Bergenia ciliata (Haw) Sternb: A Rocky Himalayan Medicinal Plant of Nepal

Bergenia ciliata (Haw) Sternb. (Saxifragaceae) is a promising, but rare and vulnerable Himalayan medicinal herb of Nepal. It grows on moist crevices of rocks and bounders. Commonly, it is known as pashanved (pasan = rock stone, ved = piercing) or rock foil. It grows between the rocks possessing lithotropic property. A decoction of rhizomes and roots of this species is used in the Ayurveda, Unani and folk systems of medicine for the treatment of ulcers, fevers, tumors, eye sores and lungs, liver, heart, urinary diseases. Due to over-exploitation for medicinal value, it is in the process of extinction. So conservation by cultivation is highly recommended. The present study was carried out on bio-guided fractionation of rhizomes powder of B. ciliata (Sternb) which is known to possess several pharmacological properties. It afforded the bioactive natural product bergenin (Isocoumarin, 3%) from ethyl acetate fraction. In addition, other four compounds namely afzelechin (2.5%), leucocyanidin, β-sitosterol and β-sitosterol glycoside were isolated from ethyl acetate fraction. co-TLC/2D-TLC, HRMS, LCMS, NMR, IR methods were employed to identify these compounds.


Introduction
B. ciliata (Haw) sternb is an evergreen perennial herb growing to 0.31 meter (1 ft) -1.0 meter (3.2 ft) by 0.5 meter (1.8ft) found in temperate Himalayas in range 400 to 4300 meter up to the frontier of vegetation. It is frost resisting and shade loving plant grows in extremely hard condition in moist stony slopes, debris, crevices, rock builders and forest shade. It is stunted creeping herb with a stout and cylindrical root stock called rhizomes (which are buff and brown outside, pinkish brown inside), stalked leaves, circular, obovate or elliptic 5-35 cm long freshly denticulate, bright green and densely ciliated at margins, sparsely hairy to glabrous on both surfaces. Its leaves are edible (pakora), used as tonic and also as plates in picnic parties and agricultural fields. It flowers and fruits in between February to August. Its beautiful pinkish white or purple flowers of cymose panicles and rounded capsules with entire bristly margins have ornamental (decorative) value as good luck (in Phool sangrum). This plant possesses urolithotropic, anticalciphication, styptic, astriangent, anti-hepatotoxic, antiinflammatory, anti-pyretic, antiviral, antimalarial, antihypertensive activity and cytoprotective effects [1], antibacterial, anti-stress, anticancer, anti-diabetic, analgesic and diuretic properties [2]. So it has strong abilities for curing respiratory-cough, pulmonary-heart, livers and urinary diseases alleviating bleeding, but intensifying immunity. Its rhizome (in the form of powder, paste or juice) is widely used in Ayurveda, Unani and other traditional/cultural system of medicines for renal disorders, oxidative stress, fever, A Rocky Himalayan Medicinal Plant of Nepal asthma and inflammation [3]. There is a good demand of rhizomes of this plant species in various pharmaceutical companies as it solubilizes stones situated around kidney, bladder and urinary tract [4]. Chemical constituents of this plant consists of many different active parameters like wax, gallicin, gallic acid, galloylepicatechin, tannins, tannic acid, mucilage, glycosides, catechin, flavonoids and other [5]. As there is no record of the research on this plant of Nepalese origin, the demonstrated significant medicinal properties prompted us to undertake phytochemical investigation. We here in report the isolation and structural elucidation of active principles.

Materials and Methods
The fresh rhizomes were randomly collected neglecting altitudinal variation from Dhaulagiri zone of 2000-4300m height during August, 2005. The voucher specimen BS-134 was authenticated at the national Herbarium and plant laboratory, Godawari, Nepal.
The air dried powdered rhizomes (1.0kg) of the plant were successively extracted with methanol (3× 1000ml) at 60°C using Soxhlet apparatus. The concentrated alcoholic extract (250gm) was diluted with cold distilled water (1000ml) and defatted with petroleum ether (1.5 × 1000ml) and petrol extract was concentrated to afford petrol extract (18gm). After defatting it was successively fractionated with chloroform (1.5 × 1000ml), ethyl acetate (1.5 × 1000ml) and methanol (1.5 × 1000ml). These extracts were concentrated under reduced pressure in Rota evaporator of Buchi type to yield chloroform extract (25gm), viscous ethyl acetate (100gm) and methanol (30gm). Brine shrimp bioassay procedure [6] and zone of inhibition test from agar cup plate method [7] (Staphylococcus aureus and Salmonella enterica) of these extracts were performed. Ethyl acetate extract (LC 50 = 1500µg/ml, high inhibition rate) found more bio active so it was chosen and fractionated by column chromatography thereafter repeated column chromatography in different solvent system of increasing polarity using pressure as a positive force. TLC, co-TLC, 2D-TLC, Prep TLC were also done during collection and identification of eluents, finally five pure compounds were isolated.
Melting points were determined on hot stage apparatus using the capillary method and are uncorrected. IR Spectra were recorded in KBr discs and nujol on a Perkin-Elmer spectrophotometer. NMR Spectra were recorded in CDCl 3 at 300 MHz on a Brunker instrument using TMS as internal standard. NMR values are in δ scales. EIMS spectra were scanned at 70 ev on a Joel D-300 instrument. Silica gel (60-120 mesh) and silica gel G were used for performing column chromatography, TLC and Prep TLC respectively. These compounds were identified on the basis of the comparison of their physical and spectral data with literature values as well as their Mass, IR and NMR spectra. The purity of these compounds was checked on TLC Silica gel UV 254 recoated plates. Final compounds/fractions were checked by using Agilent 1100 series LC-MS and a low-resonance electrospray model (ESI) with UV detection at 25nm. High resolution mass spectra (HRMS) were recorded on an Agilent ESI-TOF (time of flight) using ESI (electrospray ionization) mass spectrometer in positive-ion mode.

Results and Discussions
Brine shrimp bioactive ethyl acetate fraction on repeated column chromatography gave a white crystalline prismatic compound whose LC 50 value was 83µg/ml. This compound can be considered as a potent bioactive in bioassay as evident from important therapeutic properties like anti-cancer, antiinflammatory, anti-microbial, anti-oxidant, anti-arrhythmic, anti-arthritis, analgesic properties with the healing component of Alzheimer's disease [8]. Hz) showed C-3 and C-4 protons were also in trans orientation. The multiplet at δ 2.84 showed C-2 proton. The two protons at δ 3.30-3.45 were C-16 protons. 13 C-NMR values ranging from δ 60.3 -164.4 indicated the presence of 14 carbons. The value at δ 60.3 was methoxy group and δ 164.4 was assigned as ester (COOR) group. These spectral evidences suggested the compound to be isocoumarin. Furthermore the structure was supported by mass fragmentation pattern, 208, C 10 H 8 O 5 +. as base peak and others like 152, 165, 180, 195, 222, 237 to be consistent with the reported structural features. 1 H-1 H COSY, HMBC and HMQC were also utilized for identification. To further verify the structure, two derivatives were prepared. Simple acylation reaction in presence acetic anhydride and pyridine at 0°C for 16 hr provided the penta-O-acetyl bergenin as a white solid with HRMS calculated as 561.3523 while methylation in presence of methyl iodide and potassium carbonate resulted tri-methoxy bergenin as a colored mass with HRMS found as 357.1137. Progress of the reaction was monitored via LC-MS. Therefore, the compound was identified as bergenin [9].  Hz. Thirteen different carbon were observed from 13 C-NMR spectrum. In addition, the obtained penta-nitrated and tetra-O-methylated leucocyanidin analogs from chemical modification suggested that the compound is leucocyanidin [11]. All NMR, IR and mass spectral patterns were identical with that of reported β-Sitosterol [12].  1 H NMR spectrum pattern was similar to that of βsitosterol with some additional peaks relating to a carbohydrate moiety. The multiplet at δ 4.27 Hz was assigned for the proton of C-3. Its de-shielding may be due to attachment of β-O glucoside moiety at C-3 carbon. The proton signals at δ 3.96 Hz, 4.03 Hz, 4.27 Hz, 4.52 Hz, 4.53 Hz and 5.02 Hz in the de-shielded region were assigned for respective C-5΄, C-2΄, C-3΄, C-4΄, C-6΄, and C-1΄ protons of glucoside moiety. 13 C-NMR pattern was also similar to β-sitosterol with six more peaks confirming the glucose ring. Furthermore, the structure was supported by 1 H-1 H COSY, HMBC and HMQC. All these spectral values were found to be consistent with β-Sitosterol glucoside reported previously [12].

Conclusions
It can be stated that the B. ciliata of Nepalese origin showed interesting composition of bergenin and (+) afzelechin with medicinal and ornamental applications in traditional and folk medicine. So, it served as the valuable source of these two compounds which could potentially be used in medicinal chemistry program to discover the potent drug molecule. Research work showed that the ethyl acetate extract/fraction exhibit extreme bioactivity. It is also found that the active principles are potent in bioassay. To the best of our knowledge the above five compounds were isolated from this Himalayan plant of Nepalese origin for the first time. Further work is necessary in terms of chemical and pharmacological aspect. We have obtained different mixtures which are still to be determined.