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New Phytochemical Constituent and Bioactivities of Horwoodia dicksoniae and Rumex cyprius

Background: Many plants growing in Saudi Arabia are used in folk medicine for treatment of several diseases. Objective: Information of the chemical constituents and biological activities of plants is desirable for the discovery of therapeutic agents and discovering the actual value of folkloric remedies. Materials and Methods: The compounds were isolated and purified using silica gel column chromatography and preparative high‑performance liquid chromatography‑diode array detector (HPLC‑DAD) Method. The alcoholic extracts of these plants were evaluated for biological activities. Results: Isolation and characterization of 1‑feruloyl‑  ‑D‑glucopyranoside (1) as well as new secondary metabolite tryptophan methyl ester (2) were isolated for the 1 st time from the Horwoodia dicksoniae . The three flavones were isolated from Rumex cyprius identified as isoorientin (3), vitexin (4), and Cynarosid (5). The structures of these compounds were characterized by nuclear magnetic resonance and mass spectrometry analysis and comparing with literature. The compounds were isolated and purified using silica gel column chromatography and preparative HPLC‑DAD Method. The alcoholic extracts of these plants were evaluated for antimicrobial activities against two Gram‑positive bacteria, two Gram‑negative bacteria, and four pathogenic fungi. Both plants showed good activities against Syncephalastrum racemosum and Streptococcus pneumoniae with minimal inhibitory concentrations (MICs) 0.98 and 1.95 µ g/mL, respectively. H . dicksoniae showed good activity against Aspergillus fumigates with an MIC 1.95 µ g/mL. The two extracts showed also effective free radical scavenging activities in the 1,1‑diphenyl‑2‑picrylhydrazyl assay. H. dicksoniae exhibited remarkable cytotoxic activity against Human breast cancer mammary cancer cells‑7, Human liver cancer human hepatoma carcinoma cells‑2, and human lung carcinoma (A‑549) cell lines. Conclusions: It was suggested that further work should be carried out to isolate, purify, and characterize the active constituents responsible for the activity of these plants.


INTRODUCTION
Plant-derived substances have recently become of great interest owing to their versatile applications. Medicinal plants are the richest bioresource of drugs of traditional systems of medicine, modern medicine, and chemical precursors for synthetic drugs. Information of the chemical constituents of plants is desirable for the discovery of therapeutic agents and discovering the actual value of folkloric remedies. [1] Many plants growing in Saudi Arabia are used in folk medicine for treatment of several diseases. [2][3][4] Horwoodia dicksoniae Turrill is known in Arabic as Khuzama. Rumex genus belongs to polygonaceae family, and several species of this genus have indicated noteworthy therapeutic potentials. There are some reports in literature about evaluation of a few species of Rumex for various medicinal potential [5] Rumex cyprius revealed antibacterial activity against methicillin resistant Staphylococcus aureus, which has shown relatively high resistant when exposed to reference antibiotics. [6] In the present work, phytochemical analyses as well as antimicrobial, antioxidant, and cytotoxic activities of the alcoholic extracts of these plants were carried out.

Pharmacogn. Mag.
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MATERIALS AND METHODS Experimental
General Ultraviolet (UV) spectra were determined with a Shimadzu UV-1650PC spectrophotometer; infrared (IR) spectra were carried out on a Nicolet 205 Fourier transform IR spectrometer connected to a Hewlett-Packard ColorPro Plotter. ESIMS (positive ion acquisition mode) was carried out on an XEVO TQD triple quadruple instrument (Waters Corporation, Milford, MA 01757, USA) mass spectrometer. Proton nuclear magnetic resonance ( 1 HNMR) and 13 CNMR spectra were obtained in dimethyl sulfoxide (DMSO)-d 6

Preparation of plant extracts
The air-dried powdered herbs of H. dicksoniae (200 g) and R. cyprius (250 g) were subjected to exhaustive extraction with 70% methanol (2 L × 3 L) at room temperature. The combined methanolic extracts were concentrated under reduced pressure to dryness which gives 15 and 20 g, respectively. The concentrated methanolic extracts were suspended separately in distilled water (100 mL) and partitioned successively with n-hexane, chloroform, ethyl acetate and n-butanol to give n-hexane, chloroform, ethyl acetate, n-butanol and water fractions for each plant.

Preliminary phytochemical screening
The chemical tests were carried out on the alcoholic extract or the powdered plant material using the procedures outlined by Harborne, [7] Trease and Evans. [8] The results are presented in Table 3.

High-performance liquid chromatography-diode array detector characterization of the isolated flavones
Through comparison of different RP-HPLC retention times of compounds subfraction 3 (13.87, 14.82, and 15.94 min respectively) [ Figure 1], it was clear that no quite difference in their polarities. The UV spectra of all compounds show the same lambda maxima because the responsible chromophoric group in all compounds is the same one. The importance is the difference in the UV-absorption intensities Figure 2 between the derivatives, where it originated from the difference in the concentration of the samples.    were used for antifungal assay. The microbial species are environmental and clinically pathogenic microorganisms obtained from RCMB, Al-Azhar University. Antimicrobial tests were carried out by the agar well-diffusion method, [9,10] using 100 µL of suspension containing 1 × 10 8 colony forming units (CFU)/mL for tested bacteria and 1 × 10 4 spore/mL fungi spread on nutrient agar and malt extract agar, respectively. After the media had cooled and solidified, wells (6 mm in diameter) were made in the solidified agar and loaded with 100 µL of tested sample solutions in 1 mL DMSO with concentrations of 10 mg/mL. Negative controls were prepared using DMSO employed for dissolving the tested samples while ampicillin, gentamycin, and amphotericin B were used as positive controls for Gram-positive bacteria, Gram-negative bacteria, and fungi, respectively. The inoculated plates were then incubated for 24 h at 37°C for bacteria and 48 h at 28°C for fungi and the diameter of any resulting zones of inhibition of growth was measured in millimeter (mm). Each experiment was performed in triplicates and the data were expressed as mean ± standard deviation. The minimal inhibitory concentrations (MICs) were determined using the two-fold serial dilution technique. [11,12] The two-fold serial dilutions of the tested sample solutions were prepared. The final concentrations of the solutions were 500-0.007 µg/mL. The tubes were then inoculated with the test organisms, grown in their suitable broth for tested pathogenic bacteria (1 × 10 8 CFU/mL for bacteria and 1 × 10 4 spore/mL for fungi); each 0.5 mL received 100 µL of the above inoculum and was incubated at 37°C for 24 h for bacteria and after 48 h of incubation at 28°C for fungi. MIC values were taken as the lowest sample concentration that prevents visible bacterial growth. Each experiment was made 3 times.

Antioxidant assay
The antioxidant activity of the plant extracts was determined by the 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay. [13] Freshly prepared (0.004% w/v) methanol solution of DPPH radical was prepared and stored at 10°C in the dark. A methanol solution of the test sample was prepared. A 40 µL aliquot of the methanol solution was added to 3 mL of DPPH solution. Absorbance measurements were recorded immediately with a UV-visible spectrophotometer (Milton Roy, Spectronic 1201). The decrease in absorbance at 515 nm was determined continuously, with data being recorded at 1 min intervals until the absorbance stabilized (16 min). The absorbance of the DPPH radical without antioxidant (control) and the reference compound ascorbic acid were also measured. All the determinations were performed in three replicates and averaged. The percentage inhibition (PI) of the DPPH radical was calculated according to the formula: PI = ([(AC − AT)/AC] × 100) Where AC = absorbance of the control at t = 0 min and AT = absorbance of the sample + DPPH at t = 16 min.

Cytotoxicity assays
The plant extracts were tested for cytotoxicity against three human tumor cell lines: Human breast cancer mammary cancer cells-7 (MCF-7), Human liver cancer hepatoma carcinoma cells (HepG-2), and Human lung carcinoma (A-549) cell lines. The cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were grown on Roswell Park Memorial Institute 1640 medium (Nissui Pharm. Co., Ltd., Tokyo, Japan) supplemented with 10% inactivated fetal calf serum and 50 µg/mL gentamycin. The cells were maintained at 37°C in a humidified atmosphere with 5% CO 2 and were sub cultured 2-3 times a week. The cytotoxic activity was determined using cell viability assay method as described previously. [14,15] Percentage cell viability was calculated as the mean absorbance of control cells/mean absorbance of treated cells. Dose-response curves were prepared and the IC 50 value was determined. The results are presented in Table 2.   Data are expressed in the form of mean±SD. RCMB: Regional Center for Mycology and Biotechnology Antimicrobial unit test organisms; NA: No activity

Statistical analysis
All data were expressed as mean ± standard error of the mean. Student's t-test was applied for detecting the significance of difference between each sample; P < 0.05 was considered statistically significant. [16]

RESULTS AND DISCUSSION
The preliminary phytochemical screening of two medicinal plants, H. dicksoniae and R. cyprius, is summarized in Table 3. The results revealed the presence of medicinally active compounds in the two plants studied. Flavonoids and tannins were present in both plants. Alkaloids and saponins are present only in R. cyprius. Anthraquinones are absent from the two plants. Several studies confirmed that the presence of these important constituents contributes medicinal as well as physiological activities to the studied plants in the treatment of different diseases. Therefore, extracts from these plants can be used as a good source for valuable drugs. The H. dicksoniae and R. cyprius extract of was subjected to a succession of chromatographic procedures, including silica gel chromatography, gel permeation chromatography using Sephadex LH-20 and preparative HPLC to afford two pure isolates 1-feruloyl-β-D-glucopyranoside (1), tryptophan methyl ester (2), isoorientin (3), vitexin (4), and cynarosid (5) [ Figure 3]. The structures of the isolated compounds were established using spectroscopic analysis, especially, NMR spectra and direct comparison with published data. [17,18] The antimicrobial activities of the plant extracts were investigated in vitro against different bacteria and fungi. H. dicksoniae and R. cyprius extracts demonstrated growth inhibitory effect and variable antimicrobial activity against most of the specific organisms tested [ Table 4 and Figure 4]. They demonstrate a good activity against Aspergillus fumigatus, Streptococcus pneumoniae and E. coli. They appear to be more active against S. racemosum than the standard. They showed week antimicrobial activity against Geotricum candidum and B. subtilis. Both plant extracts did not show antimicrobial activity against the Gram-negative bacteria, P. aeruginosa and the fungus C. albicans. The results of the MIC determinations [ Table 5 and Figure 5] showed noticeable MIC values for the tested plant extracts against the entire set of the tested organisms except for P. aeruginosa and C. albicans. All the tested plant extracts exhibited both antibacterial and antifungal activities. The

The cytotoxic activity
Cytotoxic activities of the H. dicksoniae and R. cyprius extracts were

CONCLUSIONS
It was suggested that further work should be carried out to isolate, purify, and characterize the active constituents responsible for the activity of these plants. Also, additional work is encouraged to elucidate the possible mechanism of action of these constituents.