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Bioactive Chemical Constituents of Ononis natrix.

Byline: Adnan Jathlan Al-Rehaily Mohammad Shamim Ahmad Muhammad Yousaf Shabana Iqrar Khan Jamal Mustafa Babu Lal Tekwani Melissa Jacob Mohammed Abdulaziz Al-Yahya Mansour Sulaiman Al-Said Jianping Zhao and Ikhlas Ahmad Khan

Summary: Phytochemical investigation of the aerial part of Ononis natrix L. led to the isolation of a new compound 6-(2'R-acetoxypentadecyl)-2-hydroxy-4-methoxybenzoic acid (1) along with twenty one known compounds. The structures of the compounds were elucidated by standard spectroscopic methods. These compounds were screened for cytotoxic antioxidant antimicrobial antimalarial antileishmanial and antitrypanosomal activities using in vitro assays.

Keywords: Ononis natrix Leguminosae/Fabiaceae Phytomedicines Resorcinol derivatives Isocoumarins.


The majority of modern medications were derived originally from ancient herbal traditions. Medicinal plants have been used for centuries as remedies for human diseases as they contain components of therapeutic value. There are numerous natural plant products which have antifungal antibacterial and antiprotozoal activities [1]. Several plants containing volatile oils polyphenols and alkaloids as active constituents are utilized as popular folk medicines while others gained popularity in the form of finished products collectively named phytomedicines. During the second half of the 20th century the acceptance of traditional medicine as an alternative form of health care and the development of microbial resistance to the classical antibiotics led researchers to investigate the antimicrobial activity of several medicinal plants. Antimicrobial agents of plant origin have enormous therapeutic potential.

They are effective in the treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with synthetic antimicrobials [2]. Ononis natrix L. (Leguminosae/Fabiaceae) locally known as Zeetah or Rasbaa is a perennial shrublet that is distributed in Africa South-West Europe northwest region of Saudi Arabia and adjacent areas [3]. The plant is reported to have antibacterial antimicrobial [46] antihypertensive diuretic [7 8] and antirheumatic properties [9] and has been used for the treatment of urinary tract disorders [10]. A phytochemical study on O. natrix seeds [11] found that the oil was rich in fatty acids glycerids and flavonoids. In addition the phytochemical screening indicated the presence of catechic tannins saponins coumarins and terpenes. The GC analysis of the methyl esters of fatty acids showed that linoleic and linolenic acids are the most prominent constituents of the oil accounting for 33 % and 27 % respectively. O. natrix seeds are rich in proanthocyanidins (prodelphinidin and procyanidin) and flavonics aglycones the most important are quercetin kaempferol isorhamnetin nevadensin and penta-hydroxy-5673'4'-methoxy-3-flavon [11]. Flavonoids isolated from O. natrix showed significant cytotoxic activity [12]. The phytochemical composition of the extract of O. natrix has been investigated by several researchers. Feliciano et al. [10] have isolated the terpenoids sterols and three compounds: 8-hydroxy-6-methoxy-3-undecyl-34- dihydroisocoumarin; 5-(2-acetoxytridecyl)-3- methoxyphenol and 5-(2-hydroxytridecyl)-3- methoxyphenol from the hexane extract of the whole aerial part of flowering O. natrix. Canedo et al. [13] have isolated nine 5-tridecylresorcinol derivatives from the hexane extract of aerial parts of O. natrix.

Feliciano et al. [14] have isolated five 3-alkyl-34- dihydroisocoumarins and one derivative of orsellinic acid. Barrero et al. [15] have isolated N-13- docosenoylanthranilic acid (22R)-6-(2- acetoxytridecyl)-2-hydroxy-4-methoxybenzoic acid nine resorcinol derivatives eugenol and the flavone nevadensin from the acidic fraction of the hexane extract of O. natrix. Al Khalil et al [16] have isolated the N-arachidylanthranilic acid the gardenin B xanthomicrol hymenoxin 8-hydroxy-6-methoxy-3- undecyl-34-dihydroisocoumarin and medicarpin-AY- D-glucoside from the chloroform extract of the aerial parts of O. natrix. Wollenweber et al. [17] reported that more than 20 flavonoid aglycones were identified in the Mediterranean Ononis species O. fruticosa O. natrix subsp. ramosissima and O. tridentata. Barrero et al. [18] have isolated two resorcinol derivatives (RDs) 5-(2-acetoxy-8- oxotridecyl) resorcinol and 5-(2-acetoxy-7-hydroxy- 8-oxotridecyl) resorcinol -

D-glucoside betulaprenol 6 two steroids four chalcones six dihydrochalcones two flavanones and three pterocarpans from the extracts of O. natrix subsp. ramosissima. An anthranilic acid derivative from O. natrix of Jordanian origin was isolated by Nawasreh et al. [19]. The synthesis and the mass spectrometric studies of the principal dihydroisocoumarins of O. natrix were studied [20 21]. A short stereoselective synthesis of (3R)-34-dihydro-68-dimethoxy-3- undecyl-1H-[2]-benzopyran-1-one and derivatives isolated from O. natrix has been described by Saeed [22].

The present study involves the isolation of bioactive chemical constituents of aerial part of O. natrix that resulted in isolation of a new resorcinol derivative (RD) 6-(2'R-acetoxypentadecyl)-2- hydroxy-4-methoxybenzoic acid (1) (Fig. 1) ten known RDs 3-methoxy-5-tridecylphenol (3) [19 23] 5-(2'R-acetoxytridecyl)-3-methoxyphenol (6) [13 15] 5-pentadecylresorcinol (7) [24] 5- tridecylresorcinol (8) [23] 5-(2'R-acetoxytridecyl)- 13-benzenediol (9) [13 25] 5-(2'-oxotridecyl)- benzene-13-diol (10) [26] virenol C (14) [27] 6- (2'R-acetoxytridecyl)-2-hydroxy-4-methoxybenzoic acid (19) [15] 1-O-methyl-5-(2'-acetoxy-8'- hydroxytridecyl)-resorcinol (21) [13] 5-(2'-acetoxy- 8-hydroxytridecyl)-resorcinol (22) [13] six known isocoumarins 34-dihydro-8-hydroxy-6-methoxy-3- undecylisocoumarin (2) [14 25] 68-dihydroxy-3- tridecyl- 34-dihydroisocoumarin (4) [28] 34- dihydro-68-dihydroxy-3-undecylisocoumarin (5) [14]

8-hydroxy-6-methoxy-3-undecyl-34- dehydroisocoumarin (13) [14] (3R)-8-hydroxy-6- methoxy-3-(6-hydroxyundecyl)-34-dihydroiso- coumarin (15) [14] (3R)-8-hydroxy-36-dimethoxy- 3-undecyl-34-dihydroisocoumarin (16) [14] and five known flavonoids pectolinarigenin (11) [17] salvigenin (12) [28] xanthomicrol (17) [17 19] 5- demthylnobiletin (18) [29] 54'-dihydroxy-6783'- tetramethoxy flavones (20) [17 19]. Structures of these compounds (Fig. 1) were determined by comparison of their physical and spectroscopic features with those reported in the literature.

Results and Discussion

Compound 1 was isolated as a colorless gum. HRESIMS established the molecular formula of 1 as C25H40O6 (positive-ion mode m/z = 459.2746 [M + Na]+calc'd. for C25H40O6Na: 459.2710) with six degrees of unsaturation. The IR absorption bands at 3411 and 1660 cm-1 indicated the existence of hydroxyl and carbonyl groups respectively. The 1H- and 13C-NMR assignments of compound 1 were based on the COSY HMQC and HMBC spectra as well as on comparison with those previously reported for 6-(2'R-acetoxytridecyl)-2-hydroxy-4- methoxybenzoic acid (19) [15]. The 1H- and 13C- NMR spectra gave an impression of alkylresorcinol skeleton. The 13C-NMR and DEPT spectra confirmed the presence of 25 carbons consisting of two methyl one methoxy thirteen methylene three methine and six quaternary carbons. Its 13C-NMR spectrum showed six signals due to an aromatic ring 1246-

substituted (166.5 164.3 143.4 112.7 103.8 99.5) and signals of a side chain in which one carbon atom was attached to an oxygen by acetyl group ( 74.6 C- 2'). A 3H multiplet at 00.90 indicated the presence of a terminal methyl group in the side chain. The long-range HMBCs observed between H2-1' (H = 3.38 and 3.00) and C-6 (C = 143.4) C-5 (C = 112.7) C-1 (C= 103.8) C-2' (C = 74.6) revealed that the carboxylic acid unit was linked at C-1 of the aromatic ring and the acetyl group was linked at C-2' of the side chain. The 1H-1H COSY 45O spectrum of 1 indicated that the geminally coupled C-1' methylene protons (H = 3.38 and 3.00) showed vicinal couplings with the C-2' methine proton (H = 5.24) further support that the acetyl group was attached at C-2' of the side chain. All of this data corresponded to a 5-alkylresorcinol skeleton with one oxygenated function in the side chain namely acetyl group as compared to 19 in which acetyl group was also present.

However 19 have thirteen carbon side chains but 1 has fifteen carbon side chains [15]. An R absolute configuration at C-2' was proposed by comparison of the negative sign of its []D ( 14.6o) with those of similar compounds [15]. Based on the above evidence the structure of 1 was established as 6-(2'R-acetoxypentadecyl)-2-hydroxy-4- methoxybenzoic acid a new natural product and its physical and spectroscopic features were in agreement with the proposed structure (Fig. 1).

Compounds 4 and 10 were isolated for the first time from O. natrix although both of them were isolated previously from microorganisms. Compound 4 was isolated from gram-negative bacteria Ralstonia metallidurans [18] and compound 10 was isolated from actinomycetes Actinoplanes missouriensis [26]. Compounds 7 9 12 14 18 and 19 were isolated for the first time from O. natrix. The compound 13 was isolated as a natural product for the first time although earlier it was reported as a synthetic product derived from a natural product methyl-2-hydroxy-4-methoxy-6-(2'-oxotridecyl) benzoate [14]. Similarly compound 16 was reported previously as an artifact of a natural molecule (3R)- 8-hydroxy-36-dimethoxy-3-undecyl-34-dihydroiso- coumarin [14]. The presence of RD in O. natrix and several other Ononis species has been suggested to be characteristic for this genus. RD accumulates at or near the surface of diverse organs where they exhibit antibacterial antifungal and molluscicidal activities

[30 1]. Some RD mediate DNA strand scission [32 33] and act as inhibitors of glycerol-3-phosphate dehydrogenase [34]. These compounds have an oxygenation pattern in the side-chain with hydroxyl and acetoxy functions in the 2' and penultimate positions respectively. Biological Activities

These compounds were screened for antimicrobial antimalarial antileishmanial and antitrypanosomal activities using in vitro assays. Their antioxidant and cytotoxic activity was also determined. Among the resorcinol derivaties (RDs) compounds 3 69 and 19 were found to possess antimicrobial activity against various tested microorganisms. Among the six isocoumarins only one compound (5) showed antimicrobial activity while none of the flavonoids were active. As shown in Table-1 all of these compounds were active against MRSA but 8 and 9 which showed similar IC50 (1.38 and 1.35 g/mL) and MIC (2.5 g/mL) values were more potent than others. However 9 showed bactericidal activity with MBC value of 2.5 g/mL while 8 was not bactericidal. Compound 9 was also more effective than 8 against S. aureus and C. neoformans. This clearly indicates that the 13 carbon side chain in 9 enhances the activity as compared to 7 which has 15 carbon side chain.

Also 2'-acetoxyl substitution in the side chain in 9 increases the activity as compared to 8 with the unsubstituted side chain. The resorcinol derivatives 3 67 and isocoumarin 5 were also active against M. intracellulare with similar IC50 values however 5 and 7 showed bactericidal activities at 20 g/mL. Compound 19 with 13 carbon side chain showed better activity against MRSA as compared to S. aureus while compound 1 with 15 carbon side chain was inactive. This observation clearly demonstrates the importance of the 13 carbon side chain for the activity. None of the compounds showed any antimalarial activity (data not shown). Antitrypanosomal and antileishmanial activities were observed for several compounds which are shown in Table-2 in terms of IC50 and IC90 values. Two flavonoids 17 and 20 showed antitrypanosomal activity with IC50 values below 3 g/mL while RDs 3 69 and isocoumarin 5 showed IC50 values in the range of 4-8 g/mL.

The IC90 values for antitrypanosomal activity ranged from 6 to10 g/mL for these compounds. Moderate antileishmanial activity was observed for 310 13 14 and 16 with IC50 values in the range of 5-15 g/mL and IC90 values in the range of 12 36g/mL. The results indicated that some of the RDs (810) are more active than other RDs and isocoumarins. Flavonoids did not show any antileishmanial activity. These results also indicated that the two flavonoids (17 and 20) showed selectivity for trypanosoma over other organisms.

Previously reported antimicrobial activities of O. natrix [46] can be explained due to the presence of these bioactive constituents. The results from the biological activity evaluation of compounds isolated from O. natrix support the use of this plant in traditional medicine against several disorders [9 10]. The compounds were also screened for their cytotoxicity against a panel of human cancer cell lines (SK-MEL human malignant melanoma; KB human oral epidermal carcinoma; BT-549 human breast ductal carcinoma; SK-OV-3 human ovary carcinoma) and two noncancer mammalian kidney cell lines (VERO monkey kidney fibroblast and LLC-PK11 pig kidney epithelial cells). No cytotoxicity was observed against any of the cell lines up to a concentration of 10 g/mL. None of the compounds exhibited any antioxidant activity against intracellular ROS generation or cytotoxicity in HL-60 cells up to a concentration of 12.5 g/mL (data not shown).

The nontoxic nature of the constituents confirms the safety of these compounds and is in accordance with the medicinal use of this plant in humans without any health risk and toxic effects.

Table-1: Antimicrobial activity of compounds 3 5-9 and 19.

###IC50/MIC/MFC or MBC (g/mL)


###C. neoformans###S. aureus###MRSA###M. intracellulare








###Amphotericin B###0.5/1.25/1.25###NT###NT###NT


Table-2 Antitrypanosomal and antileishmanial activities of compounds 3 5-9 17 and 20

###Antitrypanosomal Activity###Antileishmanial activity



###IC90 (g/mL)


































The 1D and 2D-NMR including DQF- COSY HMQC and HMBC spectra were recorded on Bruker spectrometers operating at 400 or 500 MHz for 1H and 100 or 125 MHz for 13C. Chemical shifts were reported in parts per million (ppm) and coupling constants (J) in Hz. Proton and carbon chemical shift values are relative to the internal standard TMS. ESI-MS were obtained on an Agilent Series 1100 SL mass spectrometer. IR spectra were recorded on a Perkin-Elmer FTIR 600 series spectrometer. Column chromatography was performed using normal-phase silica gel (Merck; 230-400m). Silica gel Merck TLC Grade 7749 with gypsum binder and fluorescent indicator was used for chromatotron. HPLC was performed on a Shimadzu system (Kyoto Japan) consisting of two LC-6AD Semi-Preparative Solvent Delivery pumps coupled with Rheodyne manual injector communications bus module CBM-20A a multi wavelength photo-diode array detector (SPD-M20A)

FRC-10A fraction collector all connected to a computer system with Intel Core DUO with Microsoft XP and Shimadzu's LC solution software. It was fitted with a Shim-pack PREP-ODS (H) Kit (A) 250 mm A- 4.6 mm I.D. with 5 m particles (B) 250 mm A- 20 mm I.D. 5 m. Analytical HPLC was performed using the (A) column under gradient conditions with a mobile phase consisting of acetonitrile and water (40 : 60) programmed linearly to 100% acetonitrile over 25 min at the flow rate 1.0 mL/min. The UV detection wavelength was 254 nm. The chromatographic separation HPLC was performed using (B) column and preparative HPLC conditions were the same as those of analytical HPLC except the flow rate was 20 mL/min.

Plant Material Ononis natrix L. was collected in March 2007 from Allos Mountain Tabouk Saudi Arabia and identified by Dr. M. Yusuf taxonomist College of Pharmacy King Saud University (KSU) Riyadh Saudi Arabia. A voucher specimen (# 15756) was deposited at the herbarium of the college of Pharmacy KSU.

Extraction and Isolation

The aerial part (500 g) was briefly treated with acetone to extract the lipophilic material accumulated on the plant surfaces [17] filtered and the solution were evaporated under reduced pressure to give a viscous extract (44.6 g) of which 15 g was partitioned between n-hexane and acetonitrile (equilibrated with each other). The n-hexane and acetonitrile layers afforded 2.0 g and 10.2 g fractions respectively. The column chromatography of acetonitrile fraction (7 g) on silica gel (190 g 20 A- 4 cm) with a linear gradient elution of n-hexane-ethyl acetate to give twelve fractions (A-L). Fraction A (45.0 mg) on crystallization in n-hexane at room temperature afforded 2 (31.0 mg). Fraction B (162.8 mg) yielded 3 (77.4 mg). Fraction C (41.9 mg) on crystallization in n-hexane at room temperature afforded 4 (30.2 mg). The compound 5 (27.5 mg) was isolated from fraction D (47.0 mg).

Fraction E (341.3 mg) was subjected to chromatotron (Harrison Research Serial No. 30G Made in USA) 1mm plate and eluted with toluene and ethyl acetate (99 : 1) to give 6 (58.3 mg). Fractions F (118.4mg) and G (560 mg) afforded 7 (68.3 mg) and 8 (61.7 mg) respectively. Fraction H (418mg) was subjected to silica gel column (20 g 20 A- 4 cm) and gradient elution of n-hexane-ethyl acetate to give 9 (88.0 mg) and 10 (12 mg). Fraction I (10 mg) on crystallization in n-hexane at room temperature afforded 11 (7.4 mg). HPLC of the fraction J (79.4 mg) gave 12 (1.3 mg) 13 (8.6 mg) 14 (6.7 mg) 15 (3.8 mg) and 16 (11.0 mg). Fraction K (50 mg) on crystallization in n- hexane afforded 17 (36.7 mg). Fraction L (850 mg) was treated with chloroform that yielded insoluble 18 (9.3 mg). Mother liquor of L (807 mg) was subjected to chromatotron (4 mm plate) and eluted with ethyl acetate-petroleum ether (40-60 OC) (20 : 80) to afford five (i - v) sub-fractions.

Sub-fraction i (208 mg) was treated with n-hexane and kept in a refrigerator to afford 19 (34 mg). Sub-fraction iv (310 mg) was subjected to chromatotron (2 mm plate) and eluted with methanol - chloroform (1 : 99) to give 20 (74.1 mg). Sub-fraction v was subjected to prep. HPLC that afforded 1 (2.3 mg) 21 (17.7 mg) and 22 (57 mg). 6-(2'R-acetoxypentadecyl)-2-hydroxy-4- methoxybenzoic acid (1) Colorless gum; []20 14.6o (CHCl3 c 1.12); IR (NaCl) max3411 2930 2852 1742 1660 1576 1508 1438 cm-1;1H-NMR (400 MHz CDCl3) 11.53 (1H s OH D2O exchangeable) 6.39 (1H d J = 4 Hz H-3) 6.32 (1H d J = 4 Hz H-5) 5.24 (1H br. s H-2') 3.82 (3H s OMe-4) 3.38 (1H dd J = 14.0 5.2 Hz Ha-1') 3.00 (1H dd J = 12.0 8.0 Hz Hb-1') 1.87 (3H s H-17') 1.56 (2H br s H-3') 1.201.46 (22H br s H-4'H-14') 0.90 (3H m H- 15'); 13C NMR (100 MHz CDCl3) 174.0 (COOH) 170.8 (C-16') 166.5 (C-2) 164.3 (C-4) 143.4 (C-6) 112.7 (C-5) 103.8 (C-1) 99.5 (C-3) 74.6 (C-2') 55.4

(OMe4) 41.3 (C-1') 34.5 (C-3') 31.922.7 (C- 4'C-14') 21.1 (C-17') 14.1 (C-15'); HRESIMS m/z 459.2746 [M+Na]+ (calc'd for C25H40O6Na 459.2710).

Antimicrobial Assay

Microorganisms [fungi Candida albicans (ATCC 90028) Candida krusei (ATCC 6258) Candida glabrata (ATCC 90030) Cryptococcus neoformans (ATCC 90113) and Aspergillus fumigatus (ATCC 204305) and bacteria Staphylococcus aureus (ATCC 29213) methicillin- resistant S. aureus (MRSA ATCC 33591) Escherichia coli (ATCC 35218) Pseudomonas aeruginosa (ATTCC 27853) and Mycobacterium intracellulare (ATCC 23068)] were obtained from ATCC (Manassas VA). For all organisms excluding M. intracellulare and A. fumigatus susceptibility test was performed using a modified version of the CLSI (formerly NCCLS) methods [35 36] and optical density was used to monitor growth. Media supplemented with 5% Alamar Blue (BioSource International) was utilized for growth detection of M. intracellulare [37] and A. fumigatus [38]. Concentrations that afford 50% inhibition (IC50s) relative to controls were calculated using XLfit 4.2 software (IDBS Alameda CA) using fit model 201 based on duplicate readings.

Minimum fungicidal or bactericidal concentrations (MFC or MBC) were determined by removing 5 L from each clear well transferring to agar and incubating until growth was seen. Drug controls ciprofloxacin (ICN Biomedicals 99.3% purity) for bacteria and amphotericin B (ICN Biomedicals 94.8% purity) for fungi were included in each assay.

Antitrypanosomal Activity Assay

Blood stage trypomastigote forms of Trypanosomabrucei were cultured in HMI-9 medium supplemented with 10% fetal bovine serum at 37 oC and 5% CO2. A two days' old culture in the exponential phase was diluted with HMI-9 to 5000 cells/ml. The compounds were screened for antitrypanosomal activity according to the method described earlier [39]. The assay was set up in clear 96 well micro plates. Each well received 190 l of the culture and 10 uL of the test compound diluted in HMI-9 medium and plates were incubated for 48 h. Alamar blue (Biosource International Camarillo CA) (10 uL) was added to each well and the plates were incubated overnight at 37 oC and 5% CO2. Standard fluorescence was measured on a Fluostar Galaxy fluorometer (BMG Lab Technologies) at 544 nm ex 590 nm em. Pentamidine and a- difluoromethylornithine (DFMO) were tested as standard drugs. IC50 and IC90 values were computed by using XLfit as described above.

Antileishmanial Activity Assay

Antileishmanial activity was tested in vitro on a culture of Leishmania donovani promastigotes in a 96-well plate assay as reported earlier [40]. Compounds at various concentrations were added to the culture of Leishmania promastigotes (2 A- 106 cells/mL). Plates were incubated at 26oC for 72 hrs and growth of Leishmania promastigotes was determined by Almar Blue Assay. Pentamidine and amphotericin B were used as standard drugs. IC50 values were computed from growth inhibition curves.

Assay for Antioxidant Activity Inhibition of Reactive Oxygen Species (ROS) Generation

Antioxidant activity was determined in terms of the inhibition of intracellular generation of reactive oxygen species (ROS) in myelomonocytic cells (HL-60) by DCFH-DA method as described earlier [41]. HL-60 cells (ATCC) were cultured in RPMI 1640 medium with 10% FBS and antibiotics. For the assay cells were added to the wells of a 96- well plate (100000 cells/well). After treatment with test compounds for 30 min cells were stimulated with 100 ng/mL phorbol 12-myristate-13-acetate (PMA Sigma) for 30 min. DCFH-DA (Molecular Probes 5 g/mL) was added and cells were further incubated for 15 min. Levels of DCF produced were measured on a Spectramax plate reader with excitation wavelength of 485 nm and emission of 530 nm and percent decrease in DCF production was calculated compared to the vehicle control. The IC50 values were calculated from dose curves. Trolox was used as a positive control.

The cytotoxicity to HL-60 cells was also determined after incubating the cells (2 A- 104 cells/well in 225 L) with test samples for 48 h by XTT method [42]. Briefly 25 L of XTT-PMS solution (1 mg/mL XTT 25 M of PMS) was added to each well. After incubating for 4 h at 37 C absorbance was measured on a plate reader at a dual wavelength of 450 630 nm.

Assays for in Vitro Cytotoxicity

The compounds were tested for their in vitro cytotoxicity against a panel of human solid tumor cells (SK-MEL malignant melanoma; KB oral epidermal carcinoma; BT-549 breast ductal carcinoma and SK-OV-3 ovary carcinoma) as well as noncancerous kidney fibroblast (Vero) and kidney epithelial cells (LLC-PK11) [43]. All cell lines were obtained from ATCC. Cells (25000 cells/well) were seeded to the wells of 96-well plate and incubated for 24 h. Samples were added and cells were further incubated for 48 h. The number of viable cells was determined using Neutral Red assay [44]. Briefly the cells were washed with saline and incubated for 3 h with a solution of neutral red. The cells were washed again to remove extracellular dye. A solution of acidified ethanol was added to liberate the incorporated dye from viable cells and the absorbance was read at 450 nm.


This work was partially supported by Global Research Network for Medicinal Plants (GRNMP) and King Saud University. United States Department of Agriculture (USDA) Agricultural Research Service Specific Cooperative Agreement No. 58- 6408-1-603 and US Department of Defense CDMRP grant # W81XWH-09 (BLT) are also acknowledged for partial support of this work. Antifungal testing was supported by NIH NIAID Division of AIDS grant # AI27094.


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Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Geographic Code:7SAUD
Date:Dec 31, 2014
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