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Lipoxygenase and Urease Inhibition of Extracts of Polygonatum verticillatum Rhizome: Augmented by its Isolated Compound Santonin.

Byline: Haroon Khan Muhammad Saeed Murad Ali Khan Naveed Muhammad Afsar Khan Asmat Ullah and Safiullah

The present study was designed to explore the enzyme inhibitory profile of extracts of rhizome of Polygonatum verticillatum against lipoxygenase and urease. When tested against lipoxygenase ethyl acetate fraction was found the most potent (IC50: 69 g/ml) and the overall IC50 values of different extracts ranged from 69-174 g/ml. In urease assay n-butanol was the most potent fraction (IC50: 169 g/ml) while the overall IC50 values were in the range of 169-288 g/ml. Bioactivity guided chromatography led to the isolation of compound 1 which was characterized as santonin on the basis of various spectroscopic techniques. When santonin was tested against lipoxygenase and urease it showed potent inhibition of lipoxygenase (IC50: 27.4 M) but did not attenuate the urease activity. Our findings provided strong evidence for the enzyme inhibitory profile of the extracts of P. verticillatum rhizome and its isolated compound.

Thus results are consistent with the traditional use of the plant as an anti-inflammatory agent.

Keywords: Polygonatum verticillatum Rhizome Extract Lipoxygenase Urease Santonin.

Introduction Arachidonic acid metabolism through LOX pathway produces various biologically active lipids which are chief mediators of inflammation including leukotrienes. Leukotrienes are considered as potent mediators of inflammation and allergy [1 2]. The products of LOXs catalysed oxygenation (hydroperoxyeicosatetraenoic acids; HPETE hydroxyleicosatetraenoic acids; HETE) leukotrienes and lipoxins) are actually involved in the development of different disorders such as rheumatoid arthritis psoriasis asthmatic responses and glomerular nephritis. Consequently focus on the discovery of lipoxygenase inhibitors could be an ideal approach to deal with such disorders especially of natural origin being considered safer than synthetic agents. Results of our study pointed out marked lipoxygenase activity of the extracts and fractions of rhizome of the plant and thus offered a natural lipoxygenase inhibitor.

Urease (urea amidohydrolase; EC 3.5.1.5) is usually found in different bacteria fungi algae and plants. Infections induced by these bacteria such as Helicobacter pylori (H. pylori) and Proteus mirabilis usually have high urease activity. Urease is directly involved in the formation of infectious stones and the over expression of its activity contributes to the pathogenesis of urolithiasis pyelonephritis ammonia and hepatic encephalopathy hepatic coma and urinary catheter encrustation [3]. Urease catalyzes the hydrolysis of urea to produce ammonia and carbon dioxide and to protect the bacteria (H. pylori) in the acidic environment through elevation in pH. In agriculture high urease activity grounds environmental and economic problems [4]. Therefore targeting urease for treating pathogenic disorders may open a new line of treatment for infections caused by urease producing bacteria. The rhizome of the plant Polygonatum verticillatum showed reasonable potential as urease inhibitor.

The crude extract as well as its n-butanol and aqueous fractions showed strong potency against urease enzyme.

Polygonatum verticillatum (L.) All. (Nooreallam) represents the genus Polygonatum which comprises approximately 57 species of family Convallariaceae [5]. Several ethnobotanical uses of the plant are documented in literature. Pain pyrexia and burning sensation have been treated with syrup of fresh rhizome of Polygonatum verticillatum [5]. Rhizome of the plant is also used in combination with other herbs to promote urine discharge (diuretic) and to attenuate painful urination [6]. Of the various ethnobotanical uses some of them have already been rationalized scientifically [7-18]. In continuation of our exploration of different pharmacological activities of Polygonatum verticillatum herein we are presenting the enzyme inhibitory profile of the rhizome of the plant against lipoxygenase and urease in well established in vitro protocols followed by bioactivity guided isolation.

Results and Discussion

The lipoxygenase (LOX) inhibitory activity of rhizome of the plant is depicted in Fig. 1 and Table-1. It is evident from the results that the crude extract of rhizome possessed notable activity; the IC50 value was 102 g/ml. profound changes in the activity of rhizome were recorded upon fractionation. All the fractions had reasonable activity and potency except the n-hexane fraction. The ethyl acetate fraction was the most dominant in terms of potency; the IC50 value was 69 g/ml followed by n-butanol and chloroform fractions with IC50 values of 76 g/ml and 79 g/ml respectively. Nevertheless aqueous fraction possessed significant antagonistic activity against lipoxygenase (IC50 = 174 g/ml).

Table-1: In vitro quantitative inhibition of lipoxygenase by crude extract and various fractions of Polygonatum verticillatum.

The urease inhibition of rhizome of the plant is presented in Fig. 2 and Table-2. The crude extract of rhizome exhibited significant attenuation of enzyme (IC50 = 209 g/ml). When different fractions were tested for the activity the n-butanol fraction was the most potent fraction (IC50 = 169 g/ml) followed by chloroform fraction (IC50 = 197 g/ml). However the n-hexane and ethyl acetate fractions were inactive in urease inhibition assay.

Table-2: In vitro quantitative inhibition of urease by crude extract and various fractions of Polygonatum verticillatum

Bioactivity guided isolation led to the purification of compound 1 (Fig. 3) as a light yellowish powder. The compound was soluble in chloroform at room temperature. It was UV active and showed a molecular ion peak at m/z 246.1245 in HR-EI-MS. Thus molecular formula of the compound was determined as C15H18O3. 1H-NMR spectra of compound 1 showed a doublet resonating at d 6.65 (J = 10.0 Hz) due to olefinic C-1 methine proton and another doublet appeared at d 6.19 (J = 10.0 Hz) due to C-2 methine proton. Both of these protons showed cross peaks to each other in the COSY plot. A doublet was resonating at d 4.75 (J = 11.0 Hz) corresponding to C-6 methine proton which showed 1H-1H COSY correlation with C-7 methine proton resonating at d 1.77. The C-7 proton showed cross peaks with C-8 protons (d 1.98 dddd J = 13.0 5.0 5.0 5.0 Hz; d 1.65 dddd J = 12.75 12.75 12.75 3.5 Hz) and C-11 proton. The C-11 proton also had vicinal coupling with methyl protons of C-13 and

thus gave a dq at d 2.38 (J = 13.5 6.5 Hz). 13C-NMR analysis of compound 1 confirmed signals for 15 carbon atoms. These signals were resolved into three methyl two methylene five methine and five quaternary carbon atoms on the basis of DEPT experiment. The C-6 signal was resonating at d 81.3 showing that it was geminal to the lactone oxygen.

The two olefinic resonances for double bond appeared at d 154.9 and 125.7 due to C-1 and C-2 carbon atoms respectively. The carbonyl groups in the compound were confirmed by the signals resonating at d 186.2 and 177.5 due to C-3 and C-12 respectively [19 20]. Based on various spectroscopic techniques the structure of compound 1 was assigned as (6S7S10S11S)-41011-trimethyl-78910- tetrahydronaphtho[1612-b]furan-312(6H11H)-dione (santonin) which is a known compound [19 20].

Table-3: In vitro quantitative inhibition of urease and lipoxygenase by Santonin.

The compound 1 inhibited lipoxygenase it showed marked inhibition (IC50: 27.4 M). However it did not exhibit any activity against urease (Table- 3).

Experimental

General Experimental Conditions

The 1H-NMR spectra were measured in CDCl3 at 400 MHz on Bruker Avance spectrometer; none-deuterated residual solvent signal of CDCl3 was used as an internal standard. The 13C-NMR spectra were recorded in CDCl3 at 125 MHz. The other related techniques like COSY HMQC NOESY and HMBC were measured on Bruker Avance spectrometer at 400 MHz. Mass spectrometric analysis was based on electron impact mass spectrometry (EI-MS) and high resolution electron impact mass spectrometry (HR-EI-MS).

Plant Material

The whole plant Polygonatum verticillatum was collected from District Swat Khyber Pakhtunkhawa Pakistan in July-Aug 2007. The botanical identity of the plant was done by the

Taxonomy Department of PCSIR Laboratories Peshawar and a specimen with catalogue No: 9970 (PES) was deposited in the herbarium of PCSIR Laboratories Peshawar.

Plant Extraction and Fractionation

The shade dried rhizomes of Polygonatum verticillatum (8 Kg) were ground to fine powder. The powdered material was extracted by maceration with methanol at room temperature for 14 days with occasional shaking [21]. The methanol extracted residue was filtered off with a muslin cloth and the filtrate was concentrated under vacuum at low temperature (40 C) using rotary evaporator; yielded a dark greenish semisolid material (2.2 Kg 27.50% w/w).

Fractionation and Isolation

The crude methanol extract (1.6 Kg) was suspended in distilled water and sequentially fractionated with n-hexane chloroform ethyl acetate and n-butanol yielding n-hexane (258 g 16.13% w/w) chloroform (219 g 13.69% w/w) ethyl acetate (226 g 14.13% w/w) n-butanol (265 g 16.56% w/w) and aqueous (501 g 31.31% w/w) fractions.

The ethyl acetate soluble fraction (80 g) was subjected to column chromatography (silica gel). The initial polarity of mobile phase (methanol/chloroform; 0.5:99.5) was increased up to 12:88 (methanol:chloroform) and yielded 9 subtractions (M1-M9). Subfraction M3 was subjected again to chromatography over silica gel using methanol:ethyl acetate (0.5:99.5) which resulted into the isolation of santonin (1) as yellowish powder.

Lipoxygenase Inhibition Assay

The lipoxygenase (LOX) inhibition assay was conducted by using different dilutions of the crude extracts and fractions [22 23]. Type I-B (Soybean) lipoxygenase (1.13.11.12) and linoleic acid were purchased from Sigma (St. Loius MO) and were utilized without further purification. All other chemicals used were of analytical grade. 160 ml of sodium phosphate buffer 0.1 mM (at pH 7.0) 10 ml of sample solution (for crude extract and fractions) and 20 ml of LOX solution were mixed followed by incubation for 5 min at 258 C. The biochemical reaction was initiated by the addition of 10 l linoleic acid solution and substrate and the absorption change with the formation of (9Z11E)-13S)-13- hydroperoxyoctadeca-911-dienoate was followed for 10 min. The test sample and the control were dissolved in 50% aq. ethanol. All the reactions were performed in triplicate.

Baicalein was used as positive control of lipoxygenase while ethanol as negative control. The IC50 values were calculated using the EZ-Fit Enzyme Kinetics program (Perrella Scientific Inc. Amherst USA).

Table-4: NMR spectral data of santonin (1)

Urease Inhibition Assay

For the study of urease inhibitory activity standard documented assay was used [24]. Briefly reaction mixtures comprising 25 ml (40 units/ml) of enzyme solution and 55 ml of buffers containing urea (100 mM) were incubated with 5 l of the test samples at 30 C for 15 min in 96-well plates. Ammonia production was measured as a urease activity by indophenol method [25]. Final volumes were maintained as 200 l by adding 45 l phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprussside) and 70 l of alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaOCl) to each well. Using a microplate reader (Molecular Devices CA USA) the increase in absorbance was measured at 630 nm after 50 min at pH 6.8. The results (change in absorbance per min) were collected using SoftMax Pro software (Molecular Devices CA USA). Thiourea was used as the standard inhibitor of urease while DMSO as negative control. The % inhibition was calculated from the following equation:

Statistical Analysis

Results obtained from various in vitro experiments are expressed as mean values S.E.M. (standard error of measurement). Experiments were carried out in triplicate.

Conclusion

The rhizome of Polygonatum verticillatum registered significant inhibitory profile against lipoxygenase and urease; over-expression of which is involved in the patho-physiology of different diseases. Compound 1 strongly augmented the lipoxygenase inhibitory profile of the extracts but not the urease reversal. However further detailed studies are required for the isolation of other secondary metabolite(s) to provide better understanding of current show as well as lead compounds of clinical utility.

Acknowledgment

HK MS NM RG and S gratefully acknowledge the University of Peshawar Peshawar Pakistan for financial support.

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Publication:Journal of the Chemical Society of Pakistan
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Date:Oct 31, 2014
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