BIOCHEMICAL STUDIES ON BERBERIS LYCEUM ROYAL AND ANALYSIS OF ITS EXTRACTS FOR BIOACTIVITY

Abstract

Medicinal plants are major source of drugs used for the treatment of various health disorders. Berberis lyceum Royal, an indigenous plant of the North-East of Pakistan was selected to explore its medicinal value during this study. This plant has many therapeutic values and is being used against many diseases / infections by local population since centuries. B.lyceum remedies provided against swollen and sore eyes, broken bones, wounds, gonorrhea, curative piles, unhealthy ulcers, acute conjunctive, and in chronic ophthalmia. Therefore, thorough investigation was conducted for proximate analysis, fatty acid contents, metal ion analysis, isolation and purification of alkaloids. Bioactivity of crude extract for antimicrobial, antidiabetic and wound healing have been investigated in this study. Biochemical analysis of root samples of B. lyceum Royal showed the variation among different parameters, which include protein contents (4.4 – 6.24 %), crude fiber (14.96 – 16.40 %) and crude ash (3.79 – 6.99 %) on dry weight basis. No variation regarding crude fats (0.5 %) was found in any samples analyzed. The oil contents were determined by Soxhlet method and results revealed that the principal saturated and unsaturated fatty acid components of B. lyceum Royal root were Palmitic (16:0), Oleic (18:1) and Linoleic (18:2) acids. Palmitic acid (11.73 – 32.04 %), stearic acid (1.09 – 2.66 %), oleic acid (12.01 – 39.67 %), Linoleic acid (42.59 – 47.43 %) and linolenic acid (1.70 – 5.71) were found when oil was analyzed by gas chromatography-mass spectrometry. In all cases polyunsaturated fatty acids (PUFAs) were greater than monounsaturated fatty acids (MUFAs). The micro and macro elements of different samples were analyzed by atomic absorption spectrometry and flame photometer. The results showed that the higher mineral ion contents under investigation were found in Mansehra sample i.e. 599.12 μg /g, whereas Abbotabad had the lowest content, 242.63 μg/g. The total mineral ion contents was in the sequence of Mansehra> Kotlisattian> Bagh> Abbotabad. Calcium (Ca2+) was the highest, ranging from 456 to 187.33 μg/g and copper (Cu2+) was the lowest, ranging from 0.37 to 0.013 μg/g. Two alkaloids, berberine and palmatine were analyzed and quantified by TLC and HPLC and proton and carbon signals were detected in 1H and 13C -NMR spectra. The analysis of the NMR spectra of berberine and Palmatine revealed that the proton H-13 resonating as a singlet (H-13 of 1: δ 8.72; H-13 of 2: δ 8.81) could be used for quantification. The 1H NMR method used in this study was found to be simple, rapid and specific for the analysis of protoberberine alkaloids and required reference compound, apart from the internal standard, and an overall profile of the preparation was obtained directly. Using this method the content of protoberberine alkaloids can be determined in Berberis lyceum and other plant extracts in a shorter time than conventional method of HPLC. Bioactivity of crude extract and Berberine of B. lyceum Royal was evaluated for antimicrobial, antidiabetic and wound healing. For antimicrobial bioassay, root extracts of B. lyceum prepared in three different solvents, methanol, ethanol and aqueous and tested against different bacteria, fungi and yeast strains. Antimicrobial activities were assessed by using Disc diffusion method and Micro dilution assays. It was observed that all root extracts of Berberis lyceum were highly effective against different bacteria and fungi. The methanolic and ethanolic extracts have inhibited growth of microorganisms more effectively as compared to aqueous extract. The results obtained in present study indicates that root of B. lyceum contained some phytochemicals having antimicrobial activity and could be used for pharmaceutical industries for the development of new drugs required for human and animal health. The wound healing activities of the aqueous and methanol extracts of the root of B. lyceum were assessed using incision, excision and dead wound space models of wound repair in rats. After application of both extracts it was observed that the area of epithelialization increased, followed by an increase in wound contraction, skin breaking strength, tissue granulation, dry weight and hydroxyproline content. Histopathological studies of the granulation tissue also indicated that there was an increase in collagen formation in those rats treated with the methanol extract, compared with the control group animals. The methanol extract was more effective than the aqueous extract, but both showed significant results as compared to the control. The antidiabetic activity of the ethanol root extract of Berberis lyceum was compared with pure berberine in normal and alloxan-diabetic rats using similar doses of each. The purpose of the study was to investigate the effects of berberine and a whole extract of B. lyceum on blood glucose and other parameters associated with diabetes, to compare the effects of the crude extract with those of pure berberine and thus validate its use as a therapeutic agent, and finally to identify any contribution of the other components of the extract to these effects. Oral administration of 50 mg/kg of Berberis extract and berberine to normal and experimental diabetic rats produced a significant (p < 0.05) reduction in blood glucose levels from days 3 –7 days of treatment. Significant effects were also observed on the glucose tolerance, glycosylated haemoglobin, serum lipid profiles and body weight of experimental animals. Berberis extract and berberine demonstrated similar effects on all parameters measured, and although the extract was comparable in efficacy to berberine, it did not produce any effects additional to those shown by pure berberine. The results support the use of the extract in traditional medicine, and demonstrate that apart from being a highly cost- effective means of treating with berberine, as compared to root extract which is cheaper, easily available to rural community and also show no or very less adverse effects as compared to pure compound (berberine).