IN VITRO ANTIFUNGAL, ANTI-INFLAMMATORY AND CYTOTOXIC ACTIVITIES OF RUMEX ABYSSINICUS RHIZOME EXTRACT AND BIOASSAY-GUIDED ISOLATION OF CYTOTOXIC COMPOUNDS FROM RUMEX ABYSSINICUS

. Rumex abyssinicus showed strong cytotoxicity against HeLa cells (IC 50 = 22.25 μg/mL) and weak cytotoxicity against PC3 and BJ cells with percent inhibition of 58.6, 25.8 and 29.7% at 30.0 μg/mL. It showed moderate antifungal activity against Aspergillus niger with a percent growth inhibition of 55.5% at 3000 µg/mL. It also strongly inhibited oxidative burst with IC 50 value of 24.8 μg/mL. DCM (100%) and DCM: EtOAc (1:1) fractions showed strong cytotoxicity against HeLa cells, whilst pet ether: DCM (1:1) fraction showed strong cytotoxicity against PC3 cells with IC 50 values of 29.3, 26.3 and 24.3 μg/mL, respectively. Moreover, the DCM: EtOAc (1:1) fraction inhibited ROS production with IC 50 value of 18.8 μg/mL. Cytotoxic fractions afforded chrysophanol ( 1 ), physicon ( 2 ), emodin ( 3 ), citreorosein ( 4 ) and β-sitosterol ( 5 ). Among the isolated compounds, emodin ( 3 ) showed strong cytotoxicity against HeLa cells, whilst chrysphanol ( 1 ) and physicon ( 2 ) showed strong cytotoxicity against PC3 cells with IC 50 values of 8.94, 22.5, and 28.5 µM, respectively. In addition, emodin ( 3 ) and citreorosein ( 4 ) showed strong inhibition against ROS production with an IC 50 value of 16.2 and 38.2 μg/mL. The findings of this study suggest R. abyssinicus as a good candidate for cancer and inflammation management. technique. this assay, luminol is a probe activity against Aspergillus niger with growth inhibition of 55.5% at a concentration of 3 mg/mL. It also showed low antifungal activity against Trichophyton rubrum , Microsporum canis , and Fusarium lini pathogenic fungal which can carry 200.0, 80.0, and 50.0 g silica gel 60HF, respectively. Fractions collected from VLC were purified using column (50mm x50cm) which can carry 200 g silica gel 60HF. TLC was performed on pre-coated plates (Silica gel 60 F254, 230-400 mesh, Merck) and Al 2 O 3 plates. Spots were detected by observation under UV light (Vilber Lourmat). Spraying agents used were 10% cerium (IV) sulphate (Ce(SO 4 ) 2 ) and 5% KOH. Melting point (mp) was determined in capillary tube with a digital electrothermal melting point apparatus. UV-Vis spectral measurements were done on a Shimadzu UV-VIS recording spectrophotometer, UV-160, spectronic genesys spectrophotometer. The IR (KBr) spectral measurements were done on a Perkin Elmer 1600 and Pye Unicam Infrared spectrophotometer SP3-300. Electron ionized mass spectrometry (EI-MS) was carried out using JEOL-600H-1. The nuclear magnetic resonance (NMR) spectra were recorded in deuterated solvents (CDCl 3 or (CD 3 ) 2 CO) on a Bruker Avance III 400 and Avance Neo 600 MHz NMR spectrometers. All chemical shifts (δ) are reported in parts per million (ppm) with the solvent signal as a reference relative to TMS (δ = 0) as internal standard, while the coupling constants


INTRODUCTION
Rumex abyssinicus Jacq. (Polygonaceae), locally named in Amharic "mekmako", is an indigenous perennial herb, up to 3 m tall, with thick and fleshy rhizome. It is a medicinal plant that grows in tropical Africa, including Madagascar, more commonly in cultivated lands. In many countries of tropical Africa, leafs and tender shoots are consumed as vegetable. The rhizomes are used to refine butter and give it a bright yellow color [1]. Furthermore, it is also used as a cosmetic in northern Ethiopia for dying the palms of the hands and feet [2]. Traditional healers use this plant for the treatment of various disorders such as amebiasis, hemorrhoid, hepatitis, common cold, wound, hypertension, toothache, headache, blood pressure, asthma, liver disease, abdominal pain, tuberculosis, lung diseases , leprosy, fever, tumor and cancer [3][4][5][6].

Cytotoxic activity
The 80% EtOH extract of R. abyssinicus exhibited cytotoxicity against cervical cancer (HeLa) and prostate cancer (PC3) cell lines. R. abyssinicus inhibited the proliferation of HeLa and PC3 cancer cells by 58.6% and 25.8% at 30 µg/mL. However, the extract exerted less toxicity to normal cells (BJ) with a percent inhibition of 29.7% at 30 µg/mL. R. abyssinicus displayed potent cytotoxicity against HeLa cells with an IC50 value of 22.25 ± 0.7 μg/mL, but lower compared to standard drug doxorubicin (IC50 = 0.9 ± 0.14 µg/mL). In agreement with the current findings, Girma et al. (2013) reported that the 80% methanol in water rhizome extract of R. abyssinicus showed a chemopreventive potential against dimethylhydrazine induced colon carcinogenesis in rats, and suggested COX-2 inhibition by the anthraquinones in the extract could be one mechanism for the observed chemopreventive effect [6].
The extract of R. abyssinicus was further partitioned sequentially into eight fractions using different polarities ( The results in Table 1 indicated that the pet ether: DCM (1:1) and DCM (100%) fractions showed different cytotoxicity against HeLa and PC3 cells. Pet ether:DCM (1:1) fraction was found to be significantly cytotoxic to PC3 cells (IC50 = 26.3 ± 1.2 µg/mL, 64.9% inhibition at 30 µg/mL) but very weakly cytotoxic to HeLa cells (9.1% inhibition at 30 µg/mL). This finding indicated the responsible compounds/dose-response against the two cancer cells were different. On the other hand, DCM (100%) and DCM:EtOAc (1:1) eluted fractions were demonstrated to have comparable toxicity against HeLa and PC3 cancer cells. This may be due to the responsible compounds/dose-response of fractions being the same or exerting similar toxicity to HeLa and PC3 cancer cells. The presence of the same compounds in different fractions was also observed, but their quantity might be increased or decreased depending on the polarity of the compound and the eluent solvent used. An increment in percent inhibition of proliferation of cancer cells and a decrease in IC50 value of eluents indicated the cytotoxic activity of R. abyssinicus was related to responsible bioactive compounds and not to synergistic effect of chemical constituents found in crude extract. Furthermore, the secondary metabolites eluted from the column by less polar solvents were rich in steroids and anthraquinones, which are known to possess cytotoxic effects on cancer cells.

Anti-inflammatory activity
ROS are produced as by-products of normal biochemical reactions in the human body [20]. In inflammatory conditions, NADPH oxidases residing in the immune cells are activated and generate ROS in large quantities, creating an oxidative burst. Overproduction of ROS deregulates the cellular functions, which in turn enhances the inflammatory condition. Therefore, inhibition of ROS-induced oxidative burst is a potential therapeutic to prevent and manage inflammatorymediated diseases. The plant extract that suppresses the production of ROS may be of paramount importance in regulating diseases that originate from immune cell disturbances. The effect of R. abyssinicus extract, fractions, or compounds on production of intracellular ROS from serum opsonized zymosan activated whole blood phagocytes was evaluated by luminol enhanced chemiluminescence technique. In this assay, luminol is used as a probe having a low molecular weight. It goes inside the cell and detects intracellular ROS.

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The extract of R. abyssinicus significantly inhibited the production of whole blood ROS by 75.5% at 50 μg/mL with IC50 value of 24.8 ± 2.59 μg/mL. However, the inhibitory effect of extract on production of ROS from whole blood was lower compared to the standard drug ibuprofen with an IC50 value of 11.2 ± 1.9 μg/mL. In agreement with the current findings, Mulisa et al. [16] reported a dose-related anti-inflammatory activity of 80% methanol extract of the rhizomes of R. abyssinicus on carrageenan-induced mice paw edema following oral administration and attested the activity to secondary metabolites which have anti-inflammatory activity including tannins, flavonoids, steroids and anthraquinones [16]. 11.2 ± 1.9 DCM = dichloromethane, EtOAc = ethyl acetate, MeOH = methanol, H2O = water, Pet ether = petroleum ether, *= the % production of ROS inhibition was done at a concentration of 25 µg/mL. This inhibition of whole blood ROS production by the crude extracts could be due to specific phytochemicals present in them. Therefore, the crude extract of R. abyssinicus was further partitioned sequentially into eight solvents having different polarities (Table 2), and the capability of preventing the formation of ROS of each fraction was tested. Among the tested fractions, DCM: EtOAc (1:1), EtOAc:MeOH (1:1), DCM (100%), and EtOAc (100%) fractions inhibited more than 50% production of intracellular ROS from zymosan activated whole blood phagocytes by 89.8%, 79.4%, 58.8% and 53.1% at a concentration of 50.0 μg/mL, respectively. The DCM: EtOAc (1:1), and EtOAc:MeOH (1:1) fractions with IC50 values of 18.8 ± 0.9, and 26.3 ± 0.7 μg/mL were found to be strong inhibiters of ROS from human whole blood cells compared to DCM (100%) and EtOAc (100%) fractions with an IC50 value of 39.8 ± 7.2 μg/mL, and 44.1 ± 2.3 μg/mL, respectively. However, the standard drug Ibuprofen (IC50 = 11.2 ± 1.9 μg/mL) demonstrated stronger inhibitory potential on the production of ROS compared to all fractions.

Antifungal activity
The crude extract was evaluated for antifungal activity against six fungal strains; Trichophyton rubrum, Candida albicans, Aspergillus niger, Microsporum canis, Fusarium lini, Candida glabarata, and A. furrigatol ( Table 3). The disk diffusion method was used and the zones of growth inhibition of 80% ethanolic R. abyssinicus extract was measured in millimeters (mm) compared to the standard positive control, amphotericin B for Aspergillus niger and miconazole for the rest of the tested fungi, and a negative control, DMSO. The zone of inhibition measured was presented in Table 3.
R. abyssinicus extracts demonstrated moderate antifungal activity against Aspergillus niger with growth inhibition of 55.5% at a concentration of 3 mg/mL. It also showed low antifungal activity against Trichophyton rubrum, Microsporum canis, and Fusarium lini pathogenic fungal strains Aspergillus niger is a fungal strain that causes a disease called "black mold" on certain fruits, produce potent mycotoxins; and is also a cause of pathogenic allergens generally associated with lung infections in individuals with weak immune system [20]. The current findings are consistent with previous study which reported that MeOH, EtOAc and n-BuOH extracts of R. abyssinicus can be a potential source of antifungal agents [12].

Cytotoxic activity of compounds
The isolated constituents were estimated for their cytotoxic effects against human cervical cancer (HeLa) and human prostate cancer (PC3) cells. Among the tested compounds, chrysphanol (1) and physicon (2) and emodin (3) demonstrated strong cytotoxic activity against HeLa/PC3 cells with an IC50 values < 30 µM. According to the American National Cancer Institute (NCI), the compound is said to have cytotoxic activity if the IC50 value is < 30 µg/mL [26].
Anthraquinone-based compounds play a significant role in treatment of cancers by chemotherapeutics agents. They used as a core chemical template to achieve structural modifications, resulting in the development of new anthraquinone based compounds as promising anticancer agents. Some of the anthraquinone scaffold containing drugs such as doxorubicin, epirubicin, valrubicin, pixantrone, and mitoxantrone are currently in clinical use for various types of cancer treatments. Mechanistically, most of the anthraquinone-based compounds inhibit cancer progression by targeting essential cellular proteins [33].
β-Sitosterol (5) also demonstrated weak cytotoxic activity against prostate cancer PC3 cell lines. β-sitosterol (5) reported to exhibit a cytotoxic effect against HeLa cells [34]. β-Sitosterol (5) reported to exhibit anticancer properties against breast, prostate, colon, pancreatic, lung, stomach, and ovarian cancers by interfering with multiple cell signaling pathways including cell cycle and apoptosis [34][35][36]. Numerous studies have evidenced that the anticancer effect of βsitosterol (5) is related to the induction of apoptosis through blockade of multiple cell signalling mechanisms [35].

Anti-inflammatory activity of compounds
The inhibitory potential of compounds on the production of intracellular ROS from serum opsonized zymosan activated whole blood phagocytes is depicted in Table 4. The data collected revealed that among all tested compounds; emodin (3) and citreorosein (4) significantly inhibited the production of ROS with IC50 values of 16.20 ± 0.9 and 24.30 ± 1.6 µg/mL, respectively. However, the ROS production inhibition potential of emodin (3) and citreorosein (4) was lower compared to the standard drug Ibuprofen (IC50 value of 11.20 ± 1.9 µg/mL). Hang et al. [37] reported that the structure-activity relationship of emodin (3) indicates that the free hydroxyl group at position 3 of the anthraquinone nucleus plays an important role in the immunosuppressive effect [37].
Chrysophanol (1), physcion (2) and β-sitosterol (5) activated the production of intracellular ROS and increased the ROS production by 9.6, 10.1, and 22.7%. Literature report indicated a significant increase in intracellular ROS in chrysophanol (1), physicon (2), β-sitosterol (5) treated cells when compared to the control cells [38][39][40]. ROS has a crucial role in cell signaling and cellular functions. Mounting evidences suggest that an abnormal increase of ROS is often observed in cancer cells and that this biochemical feature can be exploited for selective killing of malignant cells. Although high levels of ROS contribute to carcinogenesis and other diseases related to oxidative damage, appropriate levels of ROS have been shown to be indispensable for cell survival, apoptosis, and differentiation [41]. Extracts, fractions, or compounds that have a potential to inhibit ROS-induced oxidative burst, can serve as effective anti-inflammatory agents. Therefore, these results provided evidence for the anti-inflammatory potential of R. abyssinicus extract, fractions, and isolated active compounds. Furthermore, it indicates the potential application of R. abyssinicus in the prevention and management of ROS-induced inflammatory conditions as a natural source of antiinflammatory drug candidates.

Apparatus and instrument
The compounds reported in this work were isolated using vacuum liquid chromatography (VLC) which carries 1 kg silica gel 60HF, and three sizes (big, medium, and small) column chromatography (CC) which can carry 200.0, 80.0, and 50.0 g silica gel 60HF, respectively. Fractions collected from VLC were purified using column (50mm x50cm) which can carry 200 g silica gel 60HF. TLC was performed on pre-coated plates (Silica gel 60 F254, 230-400 mesh, Merck) and Al2O3 plates. Spots were detected by observation under UV light (Vilber Lourmat). Spraying agents used were 10% cerium (IV) sulphate (Ce(SO4)2) and 5% KOH. Melting point (mp) was determined in capillary tube with a digital electrothermal melting point apparatus. UV-Vis spectral measurements were done on a Shimadzu UV-VIS recording spectrophotometer, UV-160, spectronic genesys spectrophotometer. The IR (KBr) spectral measurements were done on a Perkin Elmer 1600 and Pye Unicam Infrared spectrophotometer SP3-300. Electron ionized mass spectrometry (EI-MS) was carried out using JEOL-600H-1. The nuclear magnetic resonance (NMR) spectra were recorded in deuterated solvents (CDCl3 or (CD3)2CO) on a Bruker Avance III 400 and Avance Neo 600 MHz NMR spectrometers. All chemical shifts (δ) are reported in parts per million (ppm) with the solvent signal as a reference relative to TMS (δ = 0) as internal standard, while the coupling constants (J) are given in Hertz (Hz).

Plant collection
The rhizome of R. abyssinicus was collected in October, 2018, inside Arat Kilo Campus, Addis Ababa University, Addis Ababa, Ethiopia. This plant was identified and authenticated by a taxonomist and voucher specimen (JA-04-2019) was deposited at the National Herbarium, Department of Biology, College of Natural and Computational Sciences, Addis Ababa University, Addis Ababa, Ethiopia. Botanical names have been transcribed according to the nomenclature system used by The World Flora Online (http: //www.theplantlist. org).

Biological activities Cell lines and culture conditions
The human cervical cancer (HeLa), prostate cancer (PC3), human fibroblast (BJ) normal cells were separately cultured in Dulbecco's Modified Eagle Medium (DMEA), supplemented with 5% for HeLa and PC3 cells and 10% for BJ cells of fetal bovine serum (FBS), 100 IU/mL of penicillin, 100 µg/mL of streptomycin in 75 cm 2 flasks, and kept in 5% CO2 incubator at 37 o C.

Cytotoxic activity
The cytotoxicity assays (cell viability test) on HeLa, PC3, and BJ was performed according to microculture MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method as mentioned by Mosmann [42]. Briefly, 100 μL per well of cell solutions (6 × 10 4 cells per mL HeLa cells, 1 × 10 5 cells per mL PC3 cells, or 6× 10 4 cells per mL BJ cells), were added into 96well plate and incubated for 24 h at 37 °C. After overnight incubation, medium was removed and 200 µL of fresh medium was added with extract (30 µg/mL)/fraction (30 µg/mL)/compound (30 µM)/standard (30 µM) for 48 h. After this, 200 µL MTT (0.5 µg/mL) was added to each well and incubated further for 4 h at 37 °C. Then 100 μL DMSO was added to each well to dissolve the formazan crystal. The extent of MTT reduction to formazan within cells was calculated by measuring the absorbance at 570 nm (PC and HeLa) or 550 nm (BJ) using a microplate reader (Spectra Max Plus, Molecular Devices, CA, USA). Standard drug doxorubicin as a positive control and DMSO as a negative control was used to find the percent growth inhibition or decrease in viable cells. The IC50 value of active compounds (inhibition > 50% at 30 µg/mL) was calculated using the EZ-Fit software. The percent growth inhibition was calculated by using the following formula: where O.D. is optical density, NC is negative control and PC is positive control.

Selectivity index
High SI value (> 2) of a compound suggests selective toxicity against cancer cells, while a compound with SI value < 2 is considered to give general toxicity which can also cause cytotoxicity in normal cells [43]. Each SI value is calculated using the formula:

Anti-inflammatory activity test
Luminol-enhanced chemiluminescence assay was performed, as described by Helfand et. al. [44] with slight modifications. Briefly, 25 µL of 1:20 diluted whole blood in HBSS ++ was incubated with 25 µL of test sample (50.0 µg/mL for screening and three different concentrations, 1.0, 10.0, and 100.0 µg/mL for active samples), each in triplicate. Control wells received HBSS ++ and cells, but no test sample. Test was performed in white half area 96 well plates which were incubated at 37 ºC for 15 min in the thermostat chamber of luminometer (Lab systems, Helsinki, Finland). After incubation, 25 µL of 0.30% serum opsonized zymosan (SOZ) and 25 µL of 7 x 10 -5 M of intracellular ROS detecting probe, luminol was added into each well, except blank wells (containing only HBSS ++ ). The results were monitored as relative light units (RLU) reading, with peak and total integral values set with repeated scans at 50 s intervals, and 1 s point of measuring time. Standard used for the assay was Ibuprofen. The inhibition percentage (%) for each extract was calculated using the following formula: % Inhibition of ROS = − × 100%

Antifungal activity
The antifungal activity of extract to pathogenic fungi, i.e. trichophyton rubrum, candida albicans, aspergillus niger, microsporum canis, and candida glabarata, was determined using agar tube dilution method [45]. The fungal strains were grown in Sabouraud dextrose agar (SDA) which contained 2% maltose (pH 5.5-5.6), prepared by mixing 32.5 g of SDA in 500.0mL water and steamed to dissolve the contents. Into each screw caps tubes 4 mL media was dispensed. The tubes were autoclaved at 121 °C for 15 min. Tubes were allowed to cool to 50 °C and nonsolidified SDA was loaded with 66.6 µL of extracts pipetted from the stock solution (180.0 µg/mL) to give the final concentration of 3000 µg/mL. Then the tubes were allowed to solidify in slanting position at room temperature. Each tube was inoculated with a 4mm diameter piece of fungus removed from a mycelial plugs cut from the edge of seven day old cultures. For nonmycelial growth, an agar surface streak was employed. Media supplemented with DMSO was used as negative control and reference antifungal drug Amphotericin B for Aspergillus niger and Miconazole for the remaining tested fungal species were used as a positive control. The tubes that were incubated at 27 °C for 7 days and cultures were examined twice-weekly during incubation. Growth in the extract amended media was determined by measuring linear growth (mm) and growth inhibition calculated with reference to the negative control. The % inhibition from 0-39% considered as low, 40-59% as moderate, and 60-69% as good and above 70 as significantly active. The % inhibition of fungal growth for each extract was calculated using the following formula: % Fungal growth inhibition = 100% − linear growth in test (mm) linear growth in control (mm) × 100%

CONCLUSION
The bioassay-guided fractionation and isolation of compounds from R. abyssinicus against cancer cells resulted in isolation of cytotoxic compounds chrysphanol (1), physicon (2) and emodin (3) that may be a potential therapeutic agent in treatment of various cancers. The strong oxidative burst inhibitory potential of R. abyssinicus extract, fractions and isolated compounds emodin (3) and citreorosein (4) proved the effectiveness of R. abyssinicus and its isolates as natural alternatives to regulate various forms of pro-oxidative, immune disorders and inflammation. The present finding supports the folk medicinal value of R. abyssinicus in treatment of different aliments and provides scientific backing for utilizing them in such herbal preparations.