Medicinal plants are a rich source of biologically-active phytochemicals and have been used in tra-ditional medicine for centuries. Specific phyto-chemicals and extracts of their plant sources havethe ability to reduce the risk for chronic degener-ative diseases by induction of enzymes involvedin xenobiotic metabolism, many of which alsohave antioxidant and anti-inflammatory func-tions. One such multifunctional cytoprotectiveenzyme is NAD(P)H:quinone oxidoreductase. Inthis study, we prepared extracts of 27 Saudi Ara-bian medicinal plants which belong to 18 differ-ent plant families and tested their ability to in-duce NAD(P)H:quinone oxidoreductase in mu-rine hepatoma cells grown in microtiter platewells. In addition to the Brassicaceae, a knownsource of NAD(P)H:quinone oxidoreductase in-
ducer activity, we found substantial inducer activ-ity in extracts from the Apiaceae, Apocynaceae,and the Asteraceae families. Five out of a total ofeight active extracts are from plants which belongto the Asteraceae family. We further show that ar-temisinin, an agent which is used clinically for thetreatment of malaria, contributes but does notfully account for the inducer activity of the extractof Artemisia monosperma. In contrast to artemisi-nin, deoxyartemisinin is inactive in this assay,demonstrating the critical role of the endoperox-ide moiety of artemisinin for inducer activity.Thus, the NAD(P)H:quinone oxidoreductase in-ducer activity of extracts of some Saudi Arabianmedicinal plants indicates the presence of specificphytochemicals which have the potential to pro-tect against chronic degenerative diseases.
NAD(P)H:Quinone Oxidoreductase 1 Inducer Activityof Some Saudi Arabian Medicinal Plants
Authors Abdelaaty A. Shahat1,2, Mansour S. Alsaid1, Muhammad A. Alyahya1, Maureen Higgins3, Albena T. Dinkova-Kostova3,4
Affiliations 1 Medicinal, Aromatic & Poisonous Plants Research Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia2 Phytochemistry Department, National Research Centre, Dokki, Cairo, Egypt3 Jacqui Wood Cancer Centre, Division of Cancer Research, Medical Research Institute, University of Dundee, Dundee,Scotland, United Kingdom
4 Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Key wordsl" NQO1l" traditional healersl" Saudi Arabian medicinal
plantsl" chemoprotection
received Dec. 29, 2012revised January 31, 2013accepted February 8, 2013
CorrespondenceDr. Abdelaaty A. ShahatMedicinal, Aromatic & Poison-ous Plants Research CenterCollege of Pharmacy, King SaudUniversityKing Khaled streetRiyadh 11451Saudi ArabiaPhone: + 966537507320Fax: + [email protected]
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Introduction!
Plants have an enormous potential for the devel-opment of new drugs which has not been fully ex-plored yet. There are many approaches to searchfor biologically active principles in plants [1].Many medicinal plants have been used as dietarysupplements and in the treatment of numerousdiseases without proper knowledge of their func-tion. Although phytotherapy continues to be usedin several countries, few plants have received sci-entific or medical scrutiny. Moreover, a largenumber of medicinal plants possess some degreeof toxicity [2]. According to the World Health Or-ganization (WHO), more than 4000 million peo-ple in developing countries believe in the efficacyof plant remedies and use them regularly [3].However, the principal chemical compounds, ac-tive ingredients, mode of action and safety ofmany of these plants have not yet been exploredappropriately and require further research. In
Shahat AA et al. NAD(P)H :
terms of biodiversity, the flora of Saudi Arabiarepresents one of the richest areas in the ArabianPeninsula and comprises a very important geneticresource of crop and medicinal plants. It is esti-mated that the flora of Saudi Arabia has a largemedicinal species diversity, greater than 1200(more than 50%) out of its 2250 species. In addi-tion to its large number of endemic species, com-ponents of the flora of Saudi Arabia have elementsfrom Asia, Africa, and the Mediterranean region[4].NAD(P)H:quinone oxidoreductase (NQO1, EC1.6.99.2) is an enzyme with multiple cytoprotec-tive functions [5]. It is a widely distributed FAD-dependent flavoprotein that catalyzes the obliga-tory two-electron reduction of quinones, quinoneimines, nitroaromatics, and azo dyes by using ei-ther NADPH or NADH as the hydride donor. Thiscatalytic function of NQO1 plays an important cy-toprotective role as it diverts its electrophilic qui-none substrates from participating in one-elec-
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tron reductions that can generate semiquinones and various re-active oxygen species as a result of redox cycling, as well as fromparticipating in reactions with nucleophiles that could lead tosulfhydryl depletion. The gene encoding NQO1 is highly induci-ble [6] through transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) [7]. Importantly, induction of NQO1 corre-lates with protection against oxidative stress and against thetoxic and neoplastic effects of carcinogens in numerous biologicalsystems [8]. This finding has led to the development of a highlyquantitative and robust bioassay to screen pure compounds aswell as complex mixtures, such as plant extracts, for their abilityto induce NQO1 [9,10]. Moreover, the NQO1 bioassay has reliablypredicted the ability of numerous new agents to not only inducethe enzyme in various tissues in vivo but also to protect experi-mental animals against a range of chronic diseases such as car-diovascular disease, cancer, and neurodegenerative conditions. Aprominent example of the utility of the NQO1 bioassay is the iso-lation of the isothiocyanate sulforaphane from broccoli extracts[11] and the subsequent demonstrations of its chemoprotectiveactivities in animals and in humans [12].In a continuation of our interest in the complete inventory, chem-ical and biological evaluation of the medicinal plant resources ofSaudi Arabia under the auspices of the Medicinal, Aromatic andPoisonous Plant Research Center (MAPPRC) and the Departmentof Pharmacognosy, both of the College of Pharmacy, King SaudUniversity, Saudi Arabian plants which belong to 18 differentfamilies were tested for their ability to induce NQO1. The 8 plantsin which we found inducer activity are currently under phyto-chemical screening, including quantitative analysis using HPLCand HPTLC, in order to isolate and identify the active principals.
Materials and Methods!
Chemicals and reagentsAll general chemicals and reagents were of analytical grade andwere purchased from Sigma-Aldrich. Sulforaphane (99% pure)was from LKT laboratories, Inc. Artemisinin (98% pure) was ob-tained from Sigma. Deoxyartemisinin (99% pure) was purchasedfrom Toronto Research Chemicals.
Plant materialAll plants were collected from the Tanhat protected area exceptCleome ambliocarpa and Artemisia monosperma, which were col-lected from Um-Rugum and Aldahna, respectively, in April 2012.Ononis serrata and Achillea beibersteni were collected from Kabaand Albaha, Saudi Arabia in March 2010. The plants were identi-fied by the plant taxonomist at the Herbarium Unit. The voucherspecimens have been deposited at the Herbarium of the Facultyof Pharmacy, King Saud University, Riyadh, Saudi Arabia.
Sample preparationThe plants were collected and dried under shade. The dried sam-ples were powdered and used for solvent extraction. For extractpreparation, 100 g of dried sample was extracted twice with300mL of 80% methanol. The extracts were filtered throughWhatman No. 1 filter paper and concentrated using a rotaryevaporator under reduced pressure at 40°C. The dry extract ob-tained with each solvent was weighed. The percentage yield wasexpressed in terms of air dried weight of plant materials.
Shahat AA et al. NAD(P)H :Quinone Oxidoreductase 1… Planta Med 2013; 79: 459–
NQO1 assayMurine hepatoma (Hepa1c1c7) cells (ATCC) were maintained inα-MEM supplemented with 10% (v/v) fetal bovine serum thathad been heat- and charcoal-inactivated, and grown in a humidi-fied atmosphere at 37°C, 5% CO2. Dried extracts were redissolvedin 50% CH3OH/H2O (v/v) at a final concentration of 100mg/mL.Hepa1c1c7 cells (104 per well) were grown in 96-well plates for24 h and then exposed in 8 replicates to 8 different concentra-tions of extracts ranging from 0.8 to 100 µg/mL for 48 h. The finalconcentration of CH3OH in the cell culture medium was main-tained at 0.05% (v/v) in all wells. The specific activity of NQO1was evaluated in cell lysates using menadione as a substrate [9].The concentration which doubles the specific activity of NQO1(CD value) was used to quantify inducer potency. The well-estab-lished NQO1 inducer sulforaphane was used as a positive controlin each assay at a concentration range from 0.04 to 2.5 µM andconsistently found to give a CD value of 0.2 µM.
Results and Discussion!
We prepared methanolic (80%, v/v) extracts of 27 medicinalplants which belong to 18 different families: Apiaceae, Apocyna-ceae, Asteraceae, Boraginaceae, Brassicaceae, Caealpiniaceae,Cleomaceae, Convolvulaceae, Cucurbitaceae, Fabaceae, Lamia-ceae, Neuradaceae, Papilionaceae, Polygonaceae, Resedaceae,Rhamnaceae, Rutaceae, and Scrophulariaceae (l" Table 1), andtested their ability to induce NQO1 using the well-established in-ducer sulforaphane as a positive control (l" Fig. 1A). In each assay,sulforaphane consistently had a CD value of 0.2 µM and a maxi-mal magnitude of induction of 4.7-fold at a concentration of2.5 µM. Eight plant extracts showed substantial dose-dependentinducer activity (l" Table 2 and Fig. 1). In agreement with pre-vious studies on extracts and pure compounds isolated fromplant species that belong to the Brassicaceae family [10,13,14],the extract of Zilla spinosa (SY-187) showed inducer activity,with a CD value of 81 µg/mL and no detectable cytotoxicity(l" Fig. 1D). The extract of Ducrosia anethifolia (Apiaceae) (SY-182) was more potent, with a CD value of 32 µg/mL and a maxi-mal magnitude of induction of 2.5-fold (l" Fig. 1C). Extract SY-195 (from Rhazya stricta, Apocynaceae) was similar in potency(CD = 40 µg/mL) but higher in magnitude of induction: 3.6-foldat a concentration of 100 µg/mL (l" Fig. 1E). Interestingly, 5 ofthe total of 8 active extracts were from plants which belong tothe Asteraceae family. The most potent among all of the 27 ex-tracts tested in this study was the extract of Pulicaria crispa (As-teraceae) (SY-179), with a CD value of 3.3 µg/mL (l" Fig. 1B).However the protein concentrations in cell lysates that had beenexposed to concentrations of SY-179 greater than 25 µg/mL wasdose-dependently lower than in control cells, indicative of cyto-toxicity. The least potent was the extract from Achillea bieberstei-nii (Asteraceae) (SY-200) which only reached a CD value at thehighest concentration tested, 100 µg/mL (l" Fig. 1G). The extractfrom Anthemis deserti (SY-185, also from the Asteraceae family)showed high potency (CD = 16 µg/mL) and magnitude (4.5-fold)of induction, with no detectable cytotoxicity even at 100 µg/mL,the highest concentration tested (l" Fig. 1D). Similar in potency(CD = 14 µg/mL) and magnitude of induction (5-fold) was the ex-tract of Achillea fragrantissima (Asteraceae) (SY-191) (l" Fig. 1E).Extract SY-198 (Artemisia monosperma) (Asteraceae) had a CDvalue of 38 µg/mL and a magnitude of induction of 3.5-fold with
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Table 1 Medicinal plants used in the present study. * 80% methanol extracts prepared from the aerial parts (leaves and stems) of the plants.
Family (Plant species) (Voucher specimen) Traditional use Sample used*
(yield in %)
Index
Apiaceae (Ducrosia anethifolia) (15937) Analgesic and pain reliever for headache, backache, colic, and colds [20]. (26%) SY-182
Asteraceae (Achillea biebersteinii) (15960) Spasmolyse, cholerese, treatment of wounds and anti-inflammatoryactivities make it an important medicinal plant [22].
Asteraceae (Anthemis deserti) (15940) Herbal medicines, insecticides, and dyes, food additives, as well as animportant source in aromatic and cosmetic industries [24].
Cleomaceae (Cleome ambliocarpa) (15945) Stomachics, rubefacients, in the treatment of scabies, rheumatic, fever,inflammation, and as a hypoglycemic agent [33].
Cucurbitaceae (Citrullus colocynthis) (15954) Treatment of constipation, diabetes, edema, fever, jaundice, bacterialinfections as well as cancer [35].
(14.2%) SY-193
Fabaceae (Ononis serrata) (15925) Antibiotic, antipyretic, anti-inflammatory, antifungal, and antisepticactivities, treatment of skin and rheumatic diseases as well as gout [36].
Lamiaceae (Teucrium polium) (15961) Gastrointestinal disorders, inflammations, diabetes and rheumatism, anti-bacterial, antiulcer, hypotensive, antispasmodic, anorexic, and antipyreticagent. The plant possesses hypoglycemic and insulinotropic activities,reduces body weight, lowers high blood pressure and has hypolipidemic,antinociceptive, and antioxidant properties [38].
(11.5%) SY-201
Neuradaceae (Neurada procumbens) (15949) Diarrhea and dysentery; as well, it has been used as a tonic to increase heartand respiration functions [39].
(8.6%) SY-189
Papilionaceae (Trigonella hamosa) (15951) A condiment and seasoning in food preparations and hypoglycemic [40]. (22%) SY-190
Polygonaceae (Emex spinosa) (15955) Purgative, diuretic, a remedy for stomach disorders, dyspepsia, and colic[41].
(16.8%) SY-194
Polygonaceae (Rumex vasicanus) (15936) Treatment of pain, inflammation, bleeding, tinea, tumor, and constipationcough, headache, and fever [42].
Rhamnaceae (Ziziphus nummularia) (15947) Antibacterial, analgesic activities and as an anthelmintic [44]. (11.6%) SY-188
Rutaceae (Haplophyllum tuberculatum) (15932) Headaches and arthritis, to removewarts and freckles from the skin and alsoto treat skin discoloration, infections, and parasitic diseases [45]; it is used totreat malaria, rheumatoid arthritis, and gynecological disorders [46].
Antipyretic, febrifuge, and antibacterial, as a remedy for evening fever,mouth dryness, constipation, prurigo, furunculosis, sore throat, ulcerousstomatitis, tonsillitis, and in the treatment of cancer [47].
(12.12%) SY-196
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no detectable cytotoxicity at any of the concentrations tested(l" Fig. 1F).Artemisia monosperma is a source of the antimalarial agent arte-misinin (l" Fig. 2A). The presence of artemisinin has been de-tected in both Artemisia herba-alba (4.9% of dry weight) and Ar-temisia monosperma (3.6% of dry weight) [15]. Artemisinin-based combination therapies are now widely used in malariatreatment worldwide [16]. Artemisinin contains an unusual in-tramolecular peroxide bond. Peroxides are known inducers ofNQO1 [17]. We therefore considered that the inducer activity ofthe Artemisia monosperma (SY-198) extract (l" Fig. 1F) could be,at least in part, due to the presence of artemisinin. To examinethis possibility, we tested the inducer activity of pure artemisinin
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and found a modest dose-dependent increase of the specific ac-tivity of NQO1, with a maximal magnitude of induction of 1.5-fold at a concentration of 25 µM (l" Fig. 3). To establish the impor-tance of the endoperoxide moiety for the inducer activity of arte-misinin, we tested the ability of deoxyartemisinin, ametabolite ofartemisinin which only differs from the parent compound by thelack of the peroxide bond (l" Fig. 2B), to induce NQO1 and foundthat it is inactive in this assay (l" Fig. 3).Notably, deoxyartemisinin is also inactive as an antiparasiticagent, highlighting the endoperoxide-dependent mode of actionof artemisinin [18]. As NQO1 is a marker enzyme for a large net-work of cytoprotective proteins, its induction by artemisinin sug-gests that, in addition to being toxic to the parasite, artemisinin
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Table 2 NQO1 inducer potenciesof plant extracts.
Index Plant species CD value
(µg/mL)
Maximal magnitude of
induction (fold)
1 SY-182 Ducrosia anethifolia 32 2.5
2 SY-195 Rhazya stricta 40 3.6
3 SY-200 Achillea biebersteinii 100 2.0
4 SY-191 Achillea fragrantissima 14 5.1
5 SY-185 Anthemis deserti 16 4.5
6 SY-198 Artemisia monosperma 38 3.5
7 SY-184 Picriscy anocarpa 1.6
8 SY-179 Pulicaria crispa 3.3 3.8
9 SY-180 Rhantarium epapposum 1.5
10 SY-176 Echium arabicum 1.2
11 SY-183 Heliotropium ramosissimum 1.2
12 SY-187 Zilla spinosa 81 2.2
13 SY-178 Senna italic 1.4
14 SY-186 Cleome ambliocarpa 1.4
15 SY-192 Convolvulus prostates 1.4
16 SY-193 Citrullus colocynthis 1.7
17 SY-199 Ononis serrata 1.7
18 SY-175 Teucrium oliverianum 1.8
19 SY-201 Teucrium polium 1.6
20 SY-189 Neurada procumbens 1.3
21 SY-190 Trigonella hamosa 1.3
22 SY-194 Emex spinosa 1.3
23 SY-181 Rumex vasicanus 1.3
24 SY-197 Caylus eahexagyna 1.3
25 SY-188 Ziziphus nummularia 1.4
26 SY-177 Haplophyllum tuberculatum 1.3
27 SY-196 Scrophularia hypericifolia 1.3
28 Sulforaphane 0.2 µM 4.7
Fig. 1 NQO1 inducer activity of sulforaphane (A) and extracts of medicinalplants (B–G) used in this study. Hepa1c1c7 cells (104 per well) were grown in96-well plates for 24 h. The medium was removed and replaced with freshmedium containing sulforaphane at concentrations ranging from 0.04 to2.5 µM or extracts at concentrations ranging from 0.8 to100 µg/mL, and thecells were grown for a further 48 h. Cells were then lysed with digitonin, and
the specific activity of NQO1 was determined in the lysates using menadioneas a substrate. The concentration which doubles the specific activity of NQO1(CD value) was used to quantify inducer potency. Mean values for 8 replicatewells are shown for each data point. The standard deviation for each datapoint was < 5% of the value.
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may provide a further benefit by increasing the host cytoprotec-tive responses. The importance of this finding is supported by thefact that artemisinin-related compounds have been shown tohave anti-inflammatory, growth inhibitory, anti-angiogenic, and
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anti-metastatic effects, and various analogues of artemisinin arebeing developed as anticancer agents [19]. Moreover, both thepotency and the magnitude of induction of the extract of Artemi-sia monosperma are greater than that of pure artemisinin. In ad-
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Fig. 2 Chemical structures of artemisinin (A) and deoxyartemisinin (B).
Fig. 3 NQO1 inducer activity of artemisinin and deoxyartemisinin.Hepa1c1c7 cells (104 per well) were grown in 96-well plates for 24 h. Themedium was then removed and replaced with fresh medium containingserial dilutions of artemisinin or deoxyartemisinin that were prepared fromstock solutions in DMSO. The final concentration of DMSO in the cell cul-ture medium was 0.1% (v/v). Cells were grown in the presence of thecompounds for a further 48 h and then lysed with digitonin. The specificactivity of NQO1 was determined in the cell lysates using menadione as asubstrate. Mean values for 8 replicate wells are shown for each data point.The standard deviation for each data point was < 5% of the value.
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dition, the extract did not showany cytotoxicity at any of the con-centrations tested, whereas pure artemisinin was toxic at con-centrations greater than 25 µM. We therefore conclude that in-duction of NQO1 by the extract of Artemisia monosperma is onlypartially due to the presence of artemisinin, and other, perhapsmore potent constituents contribute to the overall inducer activ-ity.In summary, 8 out of 27 extracts of selected Saudi Arabianmedic-inal plants, 5 of which belong to the Asteraceae family, have theability to induce the cytoprotective marker enzyme NQO1.Although the specific indications for use of these plants in tradi-tional medicine are somewhat different, they all have two com-mon components in the disease pathogenesis: oxidative stressand inflammation. Therefore next, it will be important to identifythe active components of these extracts and test their ability toprotect against chronic degenerative diseases using models ofthe most common human chronic diseases such as cardiovascu-lar disease, neurodegenerative conditions, and cancer.
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Acknowledgements!
The authors are grateful for the Sponsorship of the ResearchCentre, College of Pharmacy, the Deanship of the Scientific Re-search, King Saud University, Riyadh, Saudi Arabia. We also thankResearch Councils UK and Cancer Research UK (C20953/A10270)for financial support.
This
is
Conflict of Interest!
The authors declare that they have no competing interests.
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