Bongardia chrysogonum(L.)Spach:a valuable source for nutraceutical and pharmacological industries

Sevgi Gezici,Nazim Sekeroglu

1Department of Molecular Biology and Genetics,Faculty of Science and Literature,Kilis 7 Aralik University,Kilis 79000,Turkey.2Advanced Technology Application and Research Center,Kilis 7 Aralik University,Kilis 79000,Turkey.3Department of Horticulture,Faculty of Agriculture,Kilis 7 Aralik University,Kilis 79000,Turkey.

Abstract Background: Bongardia chrysogonum(L.)Spach,also known as “Uruf-el-Deek”,has been used by rural communities for the treatment of various diseases and disorders,owing to its wide array of secondary metabolite compositions.Methods:The current study was conducted to determine the total polyphenolic content of Bongardia chrysogonum(L.)Spach as well as its potential free radical scavenging and metal-chelating antioxidant capacities.Its leaves,stems,and tubers were separately extracted using ethanol,water,and chloroform;their antioxidant activities were analyzed using in vitro models,including DPPH,ABTS,FRAP,and CUPRAC assays.The total polyphenolic content of the extracts was spectrophotometrically measured.Results:The richest polyphenolic content of the extracts was yielded by tuber chloroform and leaf ethanol extraction(215.09 ± 0.53 mg/g extract as gallic acid equivalent and 240.74 ±1.12 mg/g extract as quercetin equivalent,respectively).Almost all of the tested extracts exhibited remarkable antioxidant activities,with the highest antioxidant capacity observed in the leaf extracts.On the other hand,the tuber ethanol extract demonstrated the weakest DPPH radical scavenging activity(% inhibition = 49.75 ± 0.38,P ＜ 0.01),while the tuber chloroform extract showed the weakest ABTS radical scavenging activity(% inhibition = 62.61± 0.06,P ＜0.05).These were closely followed by the tuber water extract(% inhibition =63.28 ± 0.65,P ＜0.01).In terms of their FRAP and CUPRAC assays,the leaf ethanol extract was determined as the best one,with absorbance values of 3.64 ± 0.01 and 2.22 ± 0.01(P ＜0.01),respectively.Conclusion: Bongardia chrysogonum(L.)Spach possesses rich polyphenolic content and strong antioxidant activities.Thus,it could be a valuable source for the pharmaceutical industry in the prevention and treatment of diseases associated with oxidative stress.

Keywords: Bongardia chrysogonum(L.)Spach;polyphenolic content;antioxidant;medicinal plants

Background

Scavenging reactive oxygen species(e.g.,superoxide anion,hydroxyl radical,and hydrogen peroxide)and reducing oxidative stress are the most efficient treatment strategies to overcome the high mortality rates of diseases associated with oxidative stress[1,2].In folk medicine,medicinal plants are known to possess numerous pharmacological actions and a tremendous curative potential for the treatment of several diseases,including oxidative stress,diabetes,hypertension,atherosclerosis,cancer,amyotrophic lateral sclerosis,ischemia,multiple sclerosis,cardiac hypertrophy,Alzheimer’s disease,and Parkinson’s disease[3-7].Owing to their natural antioxidant capacities,plant-derived compounds serve as therapeutic alternatives against diseases caused by free radicals.In this regard,medicinal plants and natural products have been gaining significant interest among researchers worldwide for the maintenance of human health and the prevention of oxidative stress.Thus,within the last decade,researchers have focused on investigating novel,plant-based,natural antioxidants with few or absent side effects and unlimited availability[8,9].

TheBongardia chrysogonum(L.)Spach(Berberidaceae),native to the eastern Mediterranean region,is represented by only one species in the flora of Turkey.This species is generally called “Uruf-el-Deek” in Arabic and locally called “catlak out” in Anatolia.It is native to the regions of North Africa(Libya),Western-temperate Asia(Cyprus,Israel,Jordan,Lebanon,Syria,and Turkey),Caucasus(Armenia,Azerbaijan,and Georgia),and Eurasia(Russian Federation and Greece),as well as distributed in the regions of Eastern Mediterranean,North America,Jordan,Afghanistan,Syria,Iran,Iraq,Azerbaijan,and Turkey.

It is a small and edible plant with tuberous and herbaceous parts that are used for medicinal purposes.In particular,its tubers have been utilized in folk remedies since ancient times[10-12].The first report on its chemical identification and biological activities was published by Alfatafta et al.in 1989;it was entitled,“An Investigation ofBongardia chrysogonum”,in theJournal of Natural Products[13].Scientific records have stated that tuber decoctions ofBongardia chrysogonum(L.)Spach have been used by rural communities around Afghanistan,Iran,Jordan,Syria,and Turkey for the treatment of epilepsy and hematological malignancies.With the development of modern medicine,tuber decoctions have been further used to treat gastrointestinal disorders,urinary tract infections,prostate hypertrophy,hypercholesterolemia,hemorrhoids,and diabetes,owing to their antidiarrheal,antimicrobial,antiulcerogenic,anticonvulsant,and antioxidative activities[10-15].In recent literature published by Gezici and Sekeroglu in 2021,they have demonstrated that the tuber of this plant exhibits in vitro neuroprotection by inhibiting cholinesterase enzymes alongside an anticancer activity against human lung,breast,and glioma cancer cells.These results could provide valuable scientific evidence contributory to the potential use ofBongardia chrysogonumin the pharmaceutical industry[16].Moreover,the tubers ofBongardia chrysogonum(L.)Spach were reported to contain rich secondary plant metabolite compositions e.g.,alkaloids(bongardamine,bongardine,N-acetyl bongardine,lupine,lupanine,and isoquinolines),phenols(benzakaline and bongardol),triterpenoids(bongardol acetate),and saponins(glucopyranosyl,arabinofuranosyl,hederagenin,hederacoside,and leontoside);therefore,the tuber of this plant may be a promising candidate for the developmentoftherapiesagainstcancer,hemorrhoids,neurodegenerative diseases,and gastrointestinal disorders[15-19].

So far,various medicinal plants using both in vitro and in vivo antioxidant,antimicrobial,anticancer,antiproliferative,anticholinesterase,etc.,studies have been proposed to conduce novel herbal products for preventing and treating cancer as well as other neurobiological diseases;these have been achieved by our research group[8,20-27].Recent studies have reported the antioxidant and anticonvulsant effects ofBongardia chrysogonum(L.)Spach tuber extracts[10,11].However,other biological activities have not yet been identified for each plant part.Therefore,we aimed to extract the aerial parts(leaf and stem,individually)as well as the tubers ofBongardia chrysogonum(L.)Spach using solvents with different polarities,including ethanol,water,and chloroform.Additionally,content analysis was conducted using radical formation(2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid)(ABTS)and 2,2-diphenyl-1-picrylhydrazyl(DPPH))and ion-reducing(ferric reducing antioxidant power(FRAP)and cupric ion-reducing capacity(CUPRAC))assays.In addition,the total phenol and flavonoid contents of each extract were analyzed spectrophotometrically using Folin-Ciocalteu phenol and aluminum chloride reagents.To the best of our knowledge,this study is the first to assess the in vitro antioxidant activities as well as to quantify the total phenolic and flavonoid content of the aerial parts and the tuber ofBongardia chrysogonum(L.)Spach.

Methods

Chemicals and equipment

ABTS(A1888,Sigma,St.Louis,MO,USA),DPPH(D9132,Sigma,Istanbul,Turkey),5,5-dithio-bis-2-nitrobenzoic acid(D8130,Sigma,Istanbul,Turkey),ethanol 95.0%pure(EtOH,493511,Sigma,Istanbul,Turkey),methanol(82762,Sigma,Istanbul,Turkey),chloroform(02487,Sigma,Istanbul,Turkey),dimethyl sulfoxide(D8418,Sigma,Istanbul,Turkey),ferric chloride(157740,Sigma,Istanbul,Turkey),trichloroaceticacid(T6399,Sigma,Istanbul,Turkey),Folin-Ciocalteu’s phenol reagent(Sigma,Istanbul,Turkey),aluminum chloride(206911,Sigma,Istanbul,Turkey),sodium acetate(S2889,Sigma,Istanbul,Turkey),sodium carbonate(S2127,Sigma,Istanbul,Turkey),potassium persulfate(216224,Sigma,Istanbul,Turkey),copper(II)chloride(751944,Sigma,Istanbul,Turkey),neocuproine(Sigma,Istanbul,Turkey),ammonium acetate(A7262,Sigma,Istanbul,Turkey),and standards,including chlorogenic acid(C3878,Sigma),gallic acid(G7384,Sigma,Istanbul,Turkey),quercetin(Q4951,Sigma,Istanbul,Turkey),ascorbic acid(A92902,Sigma,Istanbul,Turkey),and Trolox(238813,Sigma,Istanbul,Turkey),were purchased from their respective manufacturers.Additionally,sterile 24-well and 96-well enzyme-linked immunosorbent assay microplates(Multiskan™FC Microplate Photometer,Thermo Fisher Scientific Waltham,MA,USA)were used to read the spectrophotometric absorbance.Different volumes of Eppendorf Research ® Plus Micropipette(Eppendorf AG,Hamburg,Germany)were used to perform the assays.A microplate reader spectrophotometer(Multiskan™ FC Microplate Photometer,Thermo Fisher Scientific,Waltham,MA,USA)was used to measure the absorbance.

Plant material

Various plant parts ofBongardia chrysogonum(L.)Spach were collected during spring from the Gaziantep province,Turkey.Plant materials were taxonomically identified by Res.Assist.Fatih Yayla from the Biology Department of the University of Gaziantep.A herbarium voucher(No.GAUN1603)was stored in the same department.Figure 1 shows the respective parts(aerial and tuber)of the plant used in this study;the copyright permission form for the pictures used in Figure 1 is attached as Supplementary Material.

Figure 1 Plant parts Aerial(A and B)and tuber parts(C)of Bongardia chrysogonum(L.)

Extraction of plant sample

The plant parts,including the leaves,stems,and tubers,were dried in a dark environment and powdered under laboratory conditions.Each plant part was individually extracted using the maceration method as defined in our previous publications[8,27].Briefly,plant parts were extracted using 70% ethanol,distilled water,and chloroform at room temperature for 48 hours.After filtration,the organic phases were evaporated using a rotary evaporator to obtain the crude extracts.The extraction yields for each extract were calculated,and the yields(w/w%)are presented in Table 1.The extracts were stored at-20 °C for further analyses.

Determination of total polyphenolic contents

Total phenolic contents.The total phenolic content of the extracts was quantified according to Folin-Ciocalteau’s phenol reagent method,as defined by Singleton and Rossi[28]with slight modifications[24].Accordingly,a number of dilutions of the extracts and gallic acid were prepared;the samples were mixed with 750 μL of Folin-Ciocalteu’s reagent and 600 μL of sodium carbonate in test tubes.Afterward,the tubes were incubated at 40 °C for 40 minutes.Absorbance was measured at 760 nm using a 96-well microplate reader.The total phenolic content of the extracts was expressed in terms of gallic acid equivalent(GAE)(mg/g of extract).

Total flavonoid contents.The total flavonoid content of the extracts was analyzed using the aluminum chloride colorimetric method,as defined by Woisky and Salatino with some changes[29];all the procedures and reagents were similar to those described in our previous publication[21].In this method,the dilutions from the extracts were prepared and separately mixed with 95% ethanol,0.1 mL of 10% aluminum chloride,0.1 mL of 1 mol/L sodium acetate,and 2.8 mL of distilled water.The mixture was incubated for 30 minutes at room temperature.The absorbance of the reaction mixture was then read at a wavelength of 415 nm using a 96-well microplate reader.The total flavonoid content of the extracts was expressed as quercetin equivalent(QE)( mg/g of extract).

In vitro antioxidant activity assays

The antioxidant activities of the extracts were assessed using DPPH and ABTS free radical scavenging,FRAP,and CUPRAC assays.The antioxidant potential of the extracts was determined using the concentration necessary for a 50% reduction(IC50)for each assay.

DPPH free radical scavenging capacity assay.The DPPH free radical scavenging assay was performed as described previously[8,26].Briefly,DPPH solution(0.1 mM)was prepared in 98% methanol.Different concentrations(50-1,000 μg/mL)of the extracts were mixed with 1.0 mL of DPPH solution.The mixture was then incubated in the dark for 35 minutes.The absorbance was measured at 517 nm.Ascorbic acid(1 mg/mL)was used as a standard antioxidant and was dissolved in distilled water.The inhibition of DPPH scavenging activity was calculated using the following equation;ascorbic acid was used as the reference.DPPH radical scavenging activity(%)=((1-Ac)/As)×100,where Ac is the absorbance of the reaction mixtures,including all reagents except the test sample;As is the absorbance of the reaction mixtures,including the test sample/extract.

ABTS/Trolox®-equivalentantioxidantcapacityradical scavengingactivityassay.TheABTS/Trolox®-equivalent antioxidant capacity radical cation decolorization assay was performed,as described by Nguyen et al.[30]with slight modifications.To sum up,10 mL of 2.6 mM potassium persulfate was added in 10 mL of 7.4 mM ABTS solution.The working solution was incubated for 12-15 hours in the dark at room temperature.Next,the working solution was diluted by mixing 1 mL of the stock solution into 60 mL of methanol to obtain an absorbance value of 1.1±0.02 at 734 nm.The samples were then allowed to react with the working solution for 2 hours in the dark.The percentage inhibition of the absorbance was measured at 734 nm and calculated using the following equation(Trolox was used as a reference.):ABTS scavenging effect(%)=((A-As)/A)× 100,where A is the absorbance of ABTS radical+dissolved in methanol;As is the absorbance of ABTS radical+ and test samples/extracts.

FRAP assay.FRAP analysis was accomplished with respect to previously described methods[31,32].Different concentrations of the samples were mixed with 2.5 mL each of phosphate buffer(pH 6.6)and potassium ferricyanide.Then,the mixture was incubated at 50 °C for approximately 15-20 minutes.Subsequently,trichloroacetic acid(10%)was added to the mixture and was subsequently shaken vigorously.Then,the solution was mixed with distilled water and ferric chloride(0.1% w/v)and incubated at room temperature for 30 minutes.Absorbance was measured at 700 nm using a microplate reader spectrophotometer.The increase in the absorbance of the reaction mixture corresponded to an increase in the reducing power,as compared with that of chlorogenic acid as a reference.

CUPRAC assay.The CUPRAC of the extracts was determined using the method as described by Nguyen et al.[30],with slight modifications.Briefly,1.0 mL of 10 mM copper(II)chloride solution was mixed with 1.0 mL of 7.5 mM neocuproine and 1.0 mL of 7.7%(w/v)ammonium acetate solution.Diluted extracts were then added and incubated at room temperature for 90 minutes in the dark.The absorbance of the mixture was read at 450 nm using a microplate spectrophotometer and a 96-well microplate reader,with different concentrations of Trolox as the reference.

Statistical analysis

The analyses were conducted in triplicates;data were expressed as the mean and standard deviation of the mean.Linear regression analysis was conducted to generate the IC50values.One-way analysis of variance was used to assess statistical differences between the control and sample groups.Statistical significance was set atP＜0.05,whereP＜0.01 was accepted to be very significant.

Results

Total phenol and flavonoid contents of the extracts

The total polyphenolic content of the extracts obtained from the leaves,stems,and tubers were determined spectrophotometrically(Table 1).The calibration equation for total phenol quantities of the extracts was specified according to the equation,(y = 0.9364x-0.707,r2= 0.9967),as GAE(mg/g of extract);their total flavonoid quantities were calculated according to the equation,(y = 0.9344x-0.703,r2= 0.9967),as QE(mg/g of extract).According to the data presented in Table 1,the EtOH extracts were determined to possess a higher total phenol and flavonoid content as compared with those of the water extracts.The chloroform extracts from the tubers were found to possess the highest quantity of total phenol(215.09 ± 0.53 mg/g extract as GAE),whereas the stem water extract was the poorest(122.08 ± 0.78 mg/g extract as GAE).Regarding total flavonoid amount,the leaf EtOH extract was found to exhibit the highest total flavonoid content(240.74 ± 1.12 mg/g extract,as QE),whereas the tuber water extract had the lowest(57.92±0.74 mg/g extract,as QE).In terms of total polyphenolic contents,the richest extracts were the tuber chloroform and leaf EtOH extracts(215.09 ± 0.53 mg/g extract as GAE and 240.74 ± 1.12 mg/g extract as QE,respectively)(Table 1).

Antioxidant activities of the extracts

Evaluation of the antioxidant potential of plant extracts and compounds has been the subject of extensive research,in which a number of methods have been used in previous studies.However,the antioxidant capacity of plant extracts cannot be accurately assessed using any universal method.Thus,this was tested by determining the scavenging activity of the extracts against DPPH and ABTS radicals as well as their reducing power capacity in FRAP and CUPRAC assays.Furthermore,their inhibition potentials on free radicals as well as their metal-chelating capacities are shown in Table 2.

The stem EtOH extracts showed the highest DPPH radical scavenging activity(81.75 ± 0.64% inhibition,IC50= 46.85 ± 0.19µg/mL),followed by the leaf chloroform and EtOH extracts(80.24 ±1.14% and 76.08 ± 1.02%,respectively).The tuber extracts showed a weaker scavenging effect on DPPH and ABTS radicals as compared to the leaf and stem extracts.As for ABTS radical scavenging,the EtOH leaf extracts showed the strongest scavenging activity with IC50values of 73.28 ± 0.17 µg/mL,followed by the EtOH stem extracts(IC50=91.08 ± 1.04 µg/mL)(Tables 2 and 3).

Table 1 Extraction yields,total phenol and flavonoid contents of extracts

Table 2 Radical scavenging and reducing power activities of the extracts at 1 mg/mL

Table 3 Antioxidant capacities of the extracts regarding IC50(µg/mL)values

For their reducing power capacity,the leaf EtOH extracts displayed a remarkable reducing power activity as compared to those of the other extracts and references(chlorogenic acid and Trolox).In the FRAP and CUPRAC assays,the extracts demonstrated a strong activity,of which the leaf EtOH extract possessed the highest FRAP and CUPRAC values(IC50= 121.50 ± 1.04,and 136.10 ± 0.84 µg/mL,respectively).On the other hand,the stem chloroform extract as well as the tuber water extract was revealed to have the lowest FRAP and CUPRAC values(215.81 ± 0.12 and 290.15 ± 1.38 µg/mL,respectively)(Table 3).

Discussion

Living organisms possess antioxidants to combat oxidative stress,either endogenously or exogenously produced.These substances act by inhibiting and repairing damages caused by reactive oxygen species as well as reactive nitrogen species[1,2,32].In biological systems,natural antioxidant compounds are necessary to prevent reactive oxygen species-associated reactions,including hydroxyl,superoxide,nitrous acid,nitric oxide,nitrogen dioxide,peroxyl,hypochlorous acid,hydrogen peroxide,ozone,singlet oxygen,peroxynitrite,dinitrogen trioxide,and lipid peroxide.Since free radicals and oxidants play crucial roles in the progression of chronic and degenerative diseases,such as aging,cataracts,rheumatoid arthritis,cancer,autoimmune,and neurodegenerative disorders,widespread investigations involving plant-derived antioxidants have been conducted[33-35].

Considering that our ongoing research focused on their exploration with more effectiveness and fewer side effects,the free radical scavenging activities and ion-reducing power capacities ofBongardia chrysogonum(L.)Spach extracts were analyzed.Furthermore,the total polyphenolic content of the plant was determined.To the best of our knowledge,no research has been conducted to evaluate the antioxidant activities and phytochemical properties of ethanol,aqueous,and chloroform extracts obtained from the leaves,stems,and tubers ofBongardia chrysogonum(L.)Spach.However,previous studies have demonstrated the free radical scavenging effects of the tubers ofBongardia chrysogonum(L.)Spach against DPPH.Thus,this study is the first to analyze the antioxidant activities and phytochemical properties ofBongardia chrysogonum(L.)Spach.For instance,in a recent study by Abuhamdah et al.[10],EtOH and aqueous extracts of the tuber were investigated for their antioxidant activity against DPPH and hydroxyl radicals;both extracts showed significant radical scavenging ability.In another study,the effects ofBongardia chrysogonum(L.)Spach tubers on serum total antioxidant status and serum total oxidative status were determined in the prostate tissue of streptozotocin-induced diabetes rat models;the researchers showed that the animals treated with tuber extracts had higher antioxidant status than those of the untreated control groups[13].The authors concluded that the rich phenolic and flavonoid derivatives are responsible for the high free radical scavenging and antioxidant power capacities ofBongardia chrysogonum(L.)Spach extracts.Although few studies have been carried out using the tuber part ofBongardia chrysogonum(L.)Spach,no studies have investigated the antioxidant capacities of other vegetative parts by using DPPH,ABTS,FRAP,and CUPRAC antioxidant assays.Thus,the data presented in this paper are the first to be reported in the literature.Accordingly,this study clearly showed thatBongardia chrysogonum(L.)Spach extracts have antioxidant properties.

The combination of antioxidant potential and polyphenolic contents from plant extracts may be associated with human health.It appears to effectively prevent diseases related to oxidative stress.In other words,the phenolic and flavonoid constituents of the plants correlated with the radical scavenging and reducing power capacities of the extracts.Therefore,measuring the phytochemical content in plants remains crucial for understanding their beneficial effects on human health[8,24,27,36,37].As reported in previous studies,the tubers ofBongardia chrysogonum(L.)Spach contain various phytoconstituents,such as bongardine,N-acetyl bongardine,bongardol,bongardol acetate,bongardamine etc.[15-19].Therefore,it can be assumed that the above-mentioned compounds may also be related to health promotion.

Conclusion

Based on these findings,Bongardia chrysogonum(L.)Spach may be a great source of antioxidants for the benefit of the food,medicine,pharmacology,and cosmetics industries.Furthermore,the findings of the present study could also provide valuable information for the nutraceutical and pharmacological industries;however,further studies are required to specifically identify the bioactive compounds present in the plant extracts that are deemed responsible for their antioxidant capacity.