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Extraction optimization, Total Phenolic-Flavonoids content, HPLC-DAD finger printing, antimicrobial, antioxidant and cytotoxic potentials of Chinese folklore Ephedra intermedia Schrenk & C. A. Mey

Abstract

Plants from genus Ephedra are commonly used by the Chinese people as folk medicine for treatment of various diseases. The current study was designed to explore the ethno-pharmacological based pharmacological potentials of Ephedra intermedia Schrenk & C.A. Mey. (E. intermedia). Plant aerial parts were extracted using ten solvent systems with increasing order of polarity. Samples were analyzed for total phenolic and flavonoid contents, HPLC-DAD analysis, antibacterial, antifungal, HepG2 cell line cytotoxicity, hemolysis and antioxidant potentials following standard procedures. Highest percent extract recovery was observed in Eth+WT (25.55 % w/w) solvent system. Flavonoid and phenolic contents were higher in chloroform and Met+WT fractions respectively. Considerable antibacterial activity was shown by Eth+Met extract against B. subtilis and K. pneumonia (MIC of 11.1μg/mL for each). Eth extract exhibited high antifungal activity against A. fumigates (15±0.31 mm DIZ). Met+WT extract showed significant cytotoxicity against HepG2 cell lines with IC50 of 13.51+0.69 μg/mL. Substantial free radical scavenging activity (74.9%) was observed for Met+Eth extract. In the current study, several solvent systems were used for more effective extraction of fractions and can be useful in the isolation of phytochemicals. Various fractions exhibited considerable antimicrobial, antioxidant and cytotoxic potentials. Biological potentials of E. intermedia signify its potential uses in microbial, cancer and degenerative disorders and thus warrant further detailed studies.

Keywords:
Ephedra intermedia ; Hemolytic assay; Antibacterial activity; Cancer; HepG2 cell lines

INTRODUCTION

The use of medicinal plants in drug discovery is known to human beings from time immemorial-immortal (Farnsworth, 2008Farnsworth NF. The role of ethnopharrnacology in drug development. Bioactive compounds from plants. 2008;7352.; Ayaz et al., 2017aAyaz M, Junaid M, Ullah F, Sadiq A, Shahid M, Ahmad W, et al. GC-MS analysis and gastroprotective evaluations of crude extracts, isolated saponins, and essential oil from Polygonum hydropiper L. Front Chem. 2017a;558.). Currently, plant based drugs are getting more attention in complementary and alternative medicine and as source of novel bioactive compounds (Ayaz et al., 2017bAyaz M, Junaid M, Ullah F, Subhan F, Sadiq A, Ali G, et al. Anti-Alzheimer’s Studies on β-Sitosterol Isolated from Polygonum hydropiper L. Front Pharmacol. 2017b;8697.; Ullah et al., 2018Ullah I, Subhan F, Alam J, Shahid M, Ayaz M. Suppression of cisplatin-induced vomiting by Cnnabis sativa in pigeons: neurochemical evidences. Front pharmacol. 2018;9:231.). Owing to the diversity of secondary metabolites, safety and biodegradable nature, herbal drugs may be useful alternative to the currently available drugs (Ayaz et al., 2019aAyaz M, Sadiq A, Wadood A, Junaid M, Ullah F, Khan NZ. Cytotoxicity and molecular docking studies on Phytosterols isolated from Polygonum hydropiper L. Steroids. 2019a;141:30-35.). Ephedra intermedia Schrenk & C. A. Mey belongs to the family Ephedraceae. It mainly distributes in Baluchistan, North-West Himalayas, Kashmir, western Tibet and Afghanistan (Mehra, 1950Mehra P. Occurrence of hermaphrodite flowers and the development of female gametophyte in Ephedra intermedia Shrenk et Mey. Ann Bot. 1950;14(54):165-180.). Plants from genus Ephedra are commonly used by the Chinese people as folk medicine for treatment of allergies, kidney problems, bronchial asthma, chills, diuretic, gastric and intestine inflation, fever, diaphoretic, headache, edema, flu, cold, cough, and nasal congestion (Dehkordi et al., 2015Dehkordi NV, Kachouie MA, Pirbalouti AG, Malekpoor F, Rabei M. Total phenolic content, antioxidant and antibacterial activities of the extract of Ephedra procera fisch. et mey. Acta Pol Pharm. 2015;72(2):341.).

E. intermedia is a gymnosperm shrub, belonging to the family Ephedraceae and order Gnetales. It is distributed in Balochistan, North-West Himalayas, Kashmir, western Tibet and Afghanistan (Mehra, 1950Mehra P. Occurrence of hermaphrodite flowers and the development of female gametophyte in Ephedra intermedia Shrenk et Mey. Ann Bot. 1950;14(54):165-180.). The genus Ephedra became research focus shortly after the isolation of important drug ephedrine (Abourashed et al., 2003Abourashed EA, El-Alfy AT, Khan IA, Walker L. Ephedra in perspective-a current review. Phytother Res. 2003;17(7):703-712.). Ephedrine is an alkaloid and valuable in the treatment of asthma, nasal decongestant, anti-asthmatic, promoting weight loss, cardiovascular diseases, glaucoma, narcolepsy, urinary incontinence, depression, diabetes and hyperthyroidism (Abourashed et al., 2003Abourashed EA, El-Alfy AT, Khan IA, Walker L. Ephedra in perspective-a current review. Phytother Res. 2003;17(7):703-712.; Bissa, 2015Bissa S. Evaluation of antibacterial potential of Ephedra foliata Boiss Ex CA Mey. Bioscan. 2015;10(3):1169-1172.). The genus Ephedra contains different species which are good source of medicinally important phytochemicals as shown in Figure 1 (Andraws, Chawla, Brown, 2005Andraws R, Chawla P, Brown DL. Cardiovascular effects of Ephedra alkaloids: a comprehensive review. Prog Cardiovasc Dis. 2005;47(4):217-225.). Pseudoephedrine and ephedroxane, which have anti-inflammatory potential, have been extracted from the methanolic extract of E. intermedia and Ephedra herbs (Figure 1) (Hikino et al., 1980Hikino H, Konno C, Takata H, Tamada M. Antiinflammatory principle of Ephedra herbs. Chem Pharm Bull. 1980;28(10):2900-2904.). Potential hypotensive compound flavano-flavonolephedrannin has been isolated from the roots of Ephedra plants (Hikino, Takahashi, Konno, 1982Hikino H, Takahashi M, Konno C. Structure of ephedrannin A, a hypotensive principle of Ephedra roots. Tetrahedron Letters. 1982;23(6):673-676.)a hypotensive principle of Ephedra roots. Cyclopropyl-a-amino acids have been isolated from the stem of Ephedra altissima and Ephedra foeminea (Starratt, Caveney, 1995Starratt AN, Caveney S. Four cyclopropane amino acids from Ephedra. Phytochemistry . 1995;40(2):479-481.). Ephedra species such as E. procera (Dehkordi et al., 2015Dehkordi NV, Kachouie MA, Pirbalouti AG, Malekpoor F, Rabei M. Total phenolic content, antioxidant and antibacterial activities of the extract of Ephedra procera fisch. et mey. Acta Pol Pharm. 2015;72(2):341.), E. sarcocarpa (Rustaiyan et al., 2011Rustaiyan A, Javidnia K, Farjam MH, Aboee-Mehrizi F, Ezzatzadeh E. Antimicrobial and antioxidant activity of the Ephedra sarcocarpa growing in Iran. J Med Plants Res. 2011;5(17):4251-4255.), Ephedra foliate (Bissa, 2015Bissa S. Evaluation of antibacterial potential of Ephedra foliata Boiss Ex CA Mey. Bioscan. 2015;10(3):1169-1172.), E. pachyclada and E. strobilacea (Parsaeimehr, Sargsyan, Javidnia, 2010Parsaeimehr A, Sargsyan E, Javidnia K. A comparative study of the antibacterial, antifungal and antioxidant activity and total content of phenolic compounds of cell cultures and wild plants of three endemic species of Ephedra. Molecules. 2010;15(3):1668-1678.)antifungal and antioxidant activity and total content of phenolic compounds of cell cultures and wild plants of three endemic species of Ephedra have been evaluated for their antimicrobial potential. Different species of genus Ephedra have already been evaluated for their antifungal (Wanlong, Lizhen, 1995Wanlong ZGFYD, Lizhen X. A study on antifungal activity of essential oils from Ephedra sinica and asarum heterotropoides var. Mandshuricum [J]. J Plant Prot. 1995;4014.; Bagheri-Gavkosh et al., 2009Bagheri-Gavkosh S, Bigdeli M, Shams-Ghahfarokhi M, Razzaghi-Abyaneh M. Inhibitory effects of Ephedra major host on Aspergillus parasiticus growth and aflatoxin production. Mycopathologia. 2009;168(5):249.; Mewari, Kumar, 2011Mewari N, Kumar P. Evaluation of antifungal potential of Marchantia polymorpha L., Dryopteris filix-mas (L.) Schott and Ephedra foliata Boiss. against phyto fungal pathogens. Arch Phytopathol Plant Protec. 2011;44(8):804-812.) and anticancer potential (Lee et al., 2000Lee M, Cheng B, Che C, Hsieh D. Cytotoxicity assessment of Ma-huang (Ephedra) under different conditions of preparation. Toxicol Sci. 2000;56(2):424-430.; Nam et al., 2003aNam NH, Kim HM, Bae KH, Ahn BZ. Inhibitory effects of Vietnamese medicinal plants on tube-like formation of human umbilical venous cells. Phytother Res . 2003a;17(2):107-111.; Nam et al., 2003bNam NH, Lee CW, Hong DH, Kim HM, Bae KH, Ahn BZ. Antiinvasive, antiangiogenic and antitumour activity of Ephedra sinica extract. Phytother Res . 2003b;17(1):70-76.; Takara et al., 2005Takara K, Horibe S, Obata Y, Yoshikawa E, Ohnishi N, Yokoyama T. Effects of 19 herbal extracts on the sensitivity to paclitaxel or 5-fluorouracil in HeLa cells. Biol Pharm Bull. 2005;28(1):138-142.).

FIGURE 1
Reported medicinally important phytochemicals extracted from different species of Ephedra.

Although several species of Ephedraceae family are phytochemically and pharmacologicaly explored, detailed studies on E. intermedia have not been performed yet. Consequently, this detailed study was designed to evaluate the phytochemical, antioxidant, antibacterial, antifungal, hemolytic and anticancer potential of E. intermedia. Solvents with different polarities have been used to find the best one for each type of activity. Previous studies have used one or two solvent extracts, which probably do not contain all the phytochemicals. Herein, we for the first time optimized solvent extraction system for the plant, analyzed its phytochemistry and appraised various samples for their antioxidant, anti-microbial, anti-tumor potentials and hemocompatibility behavior.

MATERIAL AND METHODS

“The Minimum Standards of Reporting Checklist contains details of the experimental design, statistics, and resources used in this study.”

Chemicals and Reagents

Analytical grade solvents were used for extraction. The solvents comprised: dimethyl sulfoxide, methanol, ethyl acetate, acetone, ethanol, chloroform, and n-hexane. The solvents and reagents like dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ferric chloride, trichloroacetic acid, ammonium molybdate, potassium ferricyanide, sulphuric acid, aluminium chloride, gallic acid, quercetin, ascorbic acid, potassium acetate, were obtained from Merck, Darmstadt, Germany, Tween-20 was bought from Merck, 2, 2-diphenyl-1-picryhydrazyl (DPPH), Folin-Ciocalteu reagent were bought from (Sigma-Aldrich) while phosphate buffer saline (PBS), nutrient agar, Sabouraud dextrose (SDA), trypton soy broth (TSB), sterile normal saline solution (0.9%), sea salt, 0.5% triton x-100,cefixime, vincristine were obtained from Sigma (Sigma Aldrich, USA) and amphotericin-B form Caisson Laboratories Hungary.

Microbial Cultures and cell line

Fungal strains include Mucor species (FCBP 0300), Aspergillus flavus (A. flavus) (FCBP 0064), Aspergillus fumigatus (A. fumigatus) (FCBP 66) and Aspergillus niger (A.niger) (FCBP 0198). The bacterial strains used as test strains for antibacterial assay were Klebsiella pneumoniae ATCC-1705 (K. pneumoniae), Staphylococcus aureus ATCC-6538 (S. aureus), Pseudomonas aeruginosa ATCC-9721 (P. aeruginosa), Staphylococcus epidermidis ATCC-12228 (S. epidermidis), Escherichia coli ATCC-25922 (E. coli) and Bacillus subtilis ATCC-6633 (B. subtilis). HepG2 cancer cell lines (RBRC-RCB1648) were used to determine the anticancer potential. All standard strains were provided by Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan. HepG2 cell line contamination was analysed following NCBI database for contamination of cell lines (Ayaz et al., 2015Ayaz M, Subhan F, Ahmed J, Khan A-u, Ullah F, Ullah I, et al. Sertraline enhances the activity of antimicrobial agents against pathogens of clinical relevance. J Biol Res-Thessalon. 2015;22(1):4.).

Collection of Plants sample

Aerial parts of Ephedra intermedia were collected in July 2016 from Kawas village of District Ziarat, Balochistan, Pakistan. Plant was identified by taxonomist Professor Dr. Zabta Khan Shinwari, and herbarium sheet under voucher no: 348 was deposited at Department of Biotechnology, Molecular Systematic and Applied Ethnobotany Lab of Quaid-i-Azam University, Islamabad, Pakistan.

Drying and extraction

The plant was shade dried for one week and ground to fine powder. Powder weighing 30 g was macerated in 150 mL of seven single solvents (WT: water, Met: methanol, Eth: ethanol, Eacet: ethyl acetate, Acet: acetone, Chlo: chloroform and NH: n-Hexane,) and three solvents in combinations (Met + WT; methanol: water, Eth + WT; ethanol: water and Met + Eth; methanol: ethanol). The ratios of methanol: water, ethanol: water and methanol: ethanol was kept as 1:1 (75 mL each) for all. The plant powder was socked for three days in shaking incubator (IRMECO Germany) and was sonicated in ultrasonic bath (Elma Schmidbauer GmbH · Gottlieb-Daimler-Str. 17 · D-78224 Singen) at 35°C for five minutes on daily basis. After three days, the solution was passed through muslin cloth to remove large debris and then filtered through Whatman No.1 filter paper. The solvent was evaporated completely from the filtrate at 40 °C in a drying oven. The crude powder extract obtained was stored at 25°C (Ayaz et al. 2017cAyaz M, Sadiq A, Junaid M, Ullah F, Subhan F, Ahmed J. Neuroprotective and anti-aging potentials of essential oils from aromatic and medicinal plants. Front. Aging Neurosci. 2017c;9168.; Kamal et al., 2017Kamal Z, Ullah F, Ahmad S, Ayaz M, Sadiq A, Imran M, et al. Saponins and solvent extracts from Atriplex laciniata L. exhibited high anthelmintic and insecticidal activities. J Tradit Chin Med. 2017;37(5):599-606.). For further experimentation, the extract was completely dissolved in 1% DMSO or the respective media used for culturing the microbes or cell lines.

Percent extract recovery of extract

The percentage extract recovery after extraction was done according to formula given below: xy x 100

Where (x) and (y) represent the weight of extract recovered and weight of plant powder used respectively (Zohra et al., 2019aZohra T, Ovais M, Khalil AT, Qasim M, Ayaz M, Shinwari ZK. Extraction optimization, total phenolic, flavonoid contents, HPLC-DAD analysis and diverse pharmacological evaluations of Dysphania ambrosioides (L.) Mosyakin & Clemants. Nat Prod Res . 2019a;33(1):136-142.).

Phytochemical analysis

Estimation of Total Flavonoid contents

Total flavonoid content was estimated according to the protocol reported by (Ul-Haq et al., 2012Ul-Haq I, Ullah N, Bibi G, Kanwal S, Ahmad MS, Mirza B. Antioxidant and cytotoxic activities and phytochemical analysis of euphorbia wallichii root extract and its fractions. Iranian J Pharma Res. 2012;11(1):241.). 20 µL of each solvent extract (4 mg/ml DMSO dilution) was taken in 96 well plate. Then 10 μL of AlCl3 (10% w/v in H2O) and 10 µl of 1.0 M CH3CO2K were added. At the end, distilled water (160 µl) was added to each well to make the final volume 200 µL and it was incubated for 30 min at 37C. Different concentrations (1.56 to 250 μg/mL) of quercetin as standard were used to obtain calibration curve (y=0.0058x+0.0538, R²=0.9925) and the results were presented as μg QE/mg plant extract (Zohra et al., 2019bZohra T, Ovais M, Khalil AT, Qasim M, Ayaz M, Shinwari ZK, et al. Bio-guided profiling and HPLC-DAD finger printing of Atriplex lasiantha Boiss. BMC Compl Alternative Med . 2019b;19(1):4.).

Estimation of Total Phenolic contents

Estimation of TPC was done by the protocol reported by (Clarke et al., 2013Clarke G, Ting KN, Wiart C, Fry J. High correlation of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric reducing activity potential and total phenolics content indicates redundancy in use of all three assays to screen for antioxidant activity of extracts of plants from the malaysian rainforest. Antioxidants. 2013;2(1):1-10.). Stock solutions (4 mg/ mL DMSO) of each solvent extracts were prepared and 20 μL of each solvent extract was added to 96 well plate followed by addition of 90μL Folin-Ciocalteu reagent (FCR). After 5 min 90 μL Na2CO3 (7.5% w/v in H2O) was added to each well and the plate was incubated for 1 h. The absorbance was measured at 760 nm using microplate reader (Bioteck, USA, Elx 800). Gallic acid and DMSO were used as positive and negative control respectively. Different concentrations of Gallic acid (1.56 -100 μg/ mL) were used to obtain calibration curve (y = 0.0427x + 0.1448, R² = 0.9826) and the results were presented as μg GAE/mg plant extract (Nasar et al., 2019Nasar MQ, Khalil AT, Ali M, Shah M, Ayaz M, Shinwari ZK. Phytochemical Analysis, Ephedra Procera CA Mey. Mediated Green Synthesis of Silver Nanoparticles, Their Cytotoxic and Antimicrobial Potentials. Medicina . 2019;55(7):369.).

High performance liquid chromatography-Diode Array detection (HPLC-DAD) analysis

HPLC-DAD analysis was performed as per our previously reported procedure from our Laboratory (Ovais et al., 2018aOvais M, Ahmad I, Khalil AT, Mukherjee S, Javed R, Ayaz M, et al. Wound healing applications of biogenic colloidal silver and gold nanoparticles: recent trends and future prospects. Appl Microbiol Biotechnol. 2018a;102(10):4305-4318.; Ahmad et al., 2020Ahmad S, Zeb A, Ayaz M, Murkovic M. Characterization of phenolic compounds using UPLC-HRMS and HPLC- DAD and anti-cholinesterase and anti-oxidant activities of Trifolium repens L. leaves. Eur Food Res Technol. 2020;246(3):485-496.).

Antioxidant Assays

Total antioxidant capacity

Phosphomolybdenum protocol was used to determine antioxidant potential of solvent extracts according to previously reported protocol (Clarke et al., 2013Clarke G, Ting KN, Wiart C, Fry J. High correlation of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric reducing activity potential and total phenolics content indicates redundancy in use of all three assays to screen for antioxidant activity of extracts of plants from the malaysian rainforest. Antioxidants. 2013;2(1):1-10.) with some modifications. 100μL of each solvent extract (4 mg/ mL in DMSO) was taken in eppendorf tubes and 900 μL of reagent (0.6 MH2SO4, 28 mM NaH2PO4(H2O) and 4 mM ammonium molybdate solution in H2O) was added. Ascorbic acid (4 mg/mL) was used as positive control and negative control contained 900 μL of reagent solution and 100 μL of DMSO without extract. All eppendorf tubes were kept at 95 °C for 90 min in water bath and then left to cool down to room temperature. After cooling, 200 μL of that reaction mixture was taken into 96 well plate and absorbance was monitor at wavelength of 630 nm by micro plate reader. Different concentrations of ascorbic acid (100, 50, 25, 12.5, 6.25, 3.12 and 1.562 μg/mL) were used to obtained calibration curve (y=0.0021x+0.099, R²=0.9802).

DPPH free radical scavenging assay

Each solvent extracts were evaluated for their free radical scavenging potential according to previously reported procedures (Clarke et al., 2013Clarke G, Ting KN, Wiart C, Fry J. High correlation of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric reducing activity potential and total phenolics content indicates redundancy in use of all three assays to screen for antioxidant activity of extracts of plants from the malaysian rainforest. Antioxidants. 2013;2(1):1-10.; Qasim et al., 2019Qasim Nasar M, Zohra T, Khalil AT, Saqib S, Ayaz M, Ahmad A, et al. Seripheidium quettense mediated green synthesis of biogenic silver nanoparticles and their theranostic applications. Green Chem Lett Rev. 2019;12(3):310-322.). DPPH solution (4.6 mg/50 mL in methanol) was prepared and 180 μL of DPPH solution was mixed with 20μL of four different dilutions of each solvent extract with final concentrations of 200, 66.66, 22.22 and 7.41 μg/mL. The reaction was incubated for 30 min at 37 °C and absorbance was recorded at 515 nm with the help of micro plate reader (Bioteck, USA, Elx 800). Ascorbic acid was used as positive control. Percent free radicals scavenging effect was determined by using the formula:

% FRSA = ( 1 - Ab s / Ab c ) × 100

Where Abs represents extract samples and Abc represents DMSO only as a negative control.

Total Reducing power assay

Total reducing power of each solvent extract was estimated by potassium ferricyanide colorimetric assay previously reported by (Jafri et al., 2017Jafri L, Saleem S, Ullah N, Mirza B. In vitro assessment of antioxidant potential and determination of polyphenolic compounds of Hedera nepalensis K. Koch. Arab J Chem. 2017;10(2):S3699-S3706.). 400 μL of phosphate buffer (pH 7,0.2 mol/L) and potassium ferricyanide (1% w/v in H2O) was mixed with 200 μL of each solvent extracts (4 mg/mL DMSO) and incubated for 20 min at 50°C. After incubation, 400 μL of trichloroacetic acid (10% w/v in H2O) was added to the mixture and centrifuged at 3000 rpm at room temperature for 10 mins. A volume of 500 μL was taken carefully from upper layer of the reaction mixture and mixed with 500 μL distilled water and 100 μL of FeCl3 (0.1% w/v in H2O). After that, 200 μL was taken from that reaction mixture into 96 well plate and absorbance was measured at 630 nm. 200 μL DMSO was used as blank for the said reaction mixture instead of extract. Different concentrations of ascorbic acid (100, 50, 25, 12.5, 6.25, 3.12 and 1.562 μg/mL) were used to obtained a calibration curve (y=0.0236x+0.0996, R2=0.9661) and the results of reducing power were expressed as μg AAE/mg extract.

Antibacterial assay

Well diffusion method was used for the antibacterial activity of each solvent extract according to protocol previously reported (Valgas et al., 2007Valgas C, Souza SMd, Smânia EF, Smânia Jr A. Screening methods to determine antibacterial activity of natural products. Braz J Microbiol. 2007;38(2):369-380.; Ayaz et al., 2016Ayaz M, Junaid M, Ullah F, Sadiq A, Ovais M, Ahmad W, et al. Chemical profiling, antimicrobial and insecticidal evaluations of Polygonum hydropiper L. BMC Compl Alternative Med. 2016;16(1):1-14.). The bacterial strains were cultured in nutrient broth and incubated in incubator (Memmert GmbH, Schwabach Germany) at 37°C for 24 h. After 24hrs, the turbidity of each inoculum was adjusted with (0.5 McFarland) standard solution. The refreshed inoculums of each strain (100 μL) was swabbed onto Tryptic soy agar plates. Each solvent extract measuring 10 µL (20 mg/ml DMSO) was added to the wells (6 mm). Cefixime and DMSO were used as positive and negative controls respectively. The plates were incubated for 24 h at 37°C. After the incubation period, the zone of clearance was observed and measured in mm with help of Vernier caliper.

Those extracts which produced inhibition zone ≥ 10 mm were consider as active and further processed to determine minimum inhibitory concentrations (MICs) by three-fold micro-broth dilution methodology (Fatima et al., 2015Fatima H, Khan K, Zia M, Ur-Rehman T, Mirza B, Haq I-u. Extraction optimization of medicinally important metabolites from Datura innoxia Mill.: an in vitro biological and phytochemical investigation. BMC Compl Alternative Med . 2015;15(1):376.). Different concentrations (100 μg/mL to 3.70 μg/mL) of active solvent extracts were prepared in 96-well microtiter plate. A stock solution of each active extract was serially diluted with Mueller Hinton broth. A standardized inoculums (5 × 104 CFU/ml) for each bacterial strain was prepared and put in 96-well microtiter plate. The plates were then kept at 37°C for an overnight incubation. The lowest concentrations inhabiting the growth were considered as MIC by measuring the OD at 600 nm and the assay was performed three times.

Antifungal assay

Disc diffusion method was used for evaluation of antifungal activity of each solvent extracts according to protocol previously described (Ihsan-Ul-Haq et al., 2012Ihsan-Ul-Haq A, Ahmed I, Hussain I, Jamil M, Mirza B. Antibacterial activity and brine shrimp toxicity of Artemisia dubia extract. Pak J Bot. 2012;44(4):1487-1490.; Zohra et al., 2019Zohra T, Ovais M, Khalil AT, Qasim M, Ayaz M, Shinwari ZK. Extraction optimization, total phenolic, flavonoid contents, HPLC-DAD analysis and diverse pharmacological evaluations of Dysphania ambrosioides (L.) Mosyakin & Clemants. Nat Prod Res . 2019a;33(1):136-142.). The fungal strains (A. niger (FCBP-01981), A. fumigatus (FCBP-66), Mucor species (FCBP-0300), and A. flavus (FCBP-0064) were obtained from fungal culture bank of Pakistan and grown on SDA media (Sabouraud Dextrose agar). The spores of each fungal strain were suspended in 0.02% Tween 20 solution and their turbidity was set according to standard (0.5 McFarland) solution. SDA plates were prepared as swabbed with 100 μL of harvested spores. Filter paper discs engrossed with 5 μL of extract (100 μg/disc) as well as 5 μL standard antifungal drug amphotericin B (250 µg/mL) and DMSO were placed on seeded SDA plates. The plates were incubated at 28°C and growth inhibition zones were measured after 24-48 h with the help of Vernier caliper.

Anti-tumoral activity

Evaluation of anticancer potential of each solvent extract against HepG2 liver cancer cell line was done according to previously reported protocol and precautions regarding the use of botanicals (Butterweck, Nahrstedt 2012Butterweck V, Nahrstedt A. What is the best strategy for preclinical testing of botanicals? A critical perspective. Planta Medica. 2012;78(08):747-754.; Ahmad et al., 2016Ahmad S, Ullah F, Ayaz M, Zeb A, Ullah F, Sadiq A. Antitumor and anti-angiogenic potentials of isolated crude saponins and various fractions of Rumexhastatus D. Don. Biol Res. 2016;491-9.). HepG2 cell line was cultured in Dulbecco’s Modified Eagle’s Medium (Lonza Bangkok 10110 Thailand) (pH=7.2) containing 10% FBS (Fetal Bovine Serum (Corning USA) in a humidified carbon dioxide incubator (37°C, 5% CO2) to attain a confluent culture. The cells (1 × 104 cells per well) were then seeded in a 96 well micro plate and incubated to adhere the cell with the walls of the wells. Thereafter, the old media was removed and fresh media (190 μL) were added into it, and 10 μL (final concentration 100 μg/mL) of each extract was added to each well. The extract solutions in final diluted form were filtered through syringe filter before adding to each well. The cells were then incubated at 37°C for 72 hr in humidified CO2 (5%) incubator. After incubation, 10 μL of sterilized MTT (Sigma Aldrich Germany) solution (1 mg/mL in distilled H2O) was added and plate was incubated again at 37°C for 4 hrs in humidified CO2 (5%) incubator. Then, 100 µL of DMSO (Sigma Aldrich Germany) was added to each well and mixed with the cells thoroughly for the complete dissolution of formazan crystals. DMSO (1%) in PBS (phosphate buffer saline) (Corning USA) and serial dilutions of vincristine was used as negative and positive control respectively. The absorbance was measured at 540 nm using a micro plate reader.

Hemolytic assay

Erythrocytes were obtained from fresh human blood by centrifuging (Memmet Germany) 1 mL of blood at 14000 rpm for 5 min. After centrifugation, the supernatant was discarded, 200 µL of precipitated erythrocytes were taken in a falcon tube and 9.8 mL of PBS was added to it. The resulting mixture was centrifuged for 10 min at 2000 rpm for washing. After centrifugation, the supernatant was discarded and washing step was repeated three times. Further, 100 µL of red blood cell suspension in PBS (4% volume) was placed in 96 well plate and 10 µL of plant extract (200 µg/mL) was added to each well. The volume was adjusted to 200 µL by adding 90 µL PBS, and the plates were incubated for one hour at 5°C. Triton X-100 (0.5%) was taken as positive control while the solvent DMSO and PBS were used as negative controls. The hemoglobin release was observed and reading was taken at 540 nm using microplate ELISA (BioTek Instruments, United States) reader. Percent hemolysis was calculated by using formula:

P e r c e n t h e m o l i s y s = ( A b s T - A b s B A b s P - A b s B ) x

Here: is the absorbance of extract sample, is absorbance of PBS (negative control) and is the absorbance of Triton X-100 (0.5%) (positive control)

Statistical analysis

Each experiment was performed at least in triplicates; presented data are as mean ± SD (standard deviation). Origin Pro 8 and GraphPad softwares were used to present data graphically.

RESULTS

Percent extract recovery of extract

Extraction of E. intermedia aerial parts was done using solvents with different polarity and the maximum % extract recovery was found in Eth + WT extract (25.55 % w/w) followed by Met extract (20.75%w/w) and Met + WT (19.65%w/w) as shown in Table I. While Chlo and Eacet extracts exhibited minimum yield (0.45%w/w and 0.80%w/w) respectively.

TABLE I
Percent extract recovery of E. intermedia using different solvents

Total Flavonoid contents

The Highest TFC was found in Chloroform and Eacet extract with flavonoid contents of 204 and 197 μg QE/mg extract respectively as shown in Figure 2.

FIGURE 2
Total Flavonoid content μg/mg extract quercetin equivalent (QE) of E. intermedia. Values obtained are as mean ± Standard error from three separate experiments.

Total phenolic contents

Figure 3 shows the total phenolic contents of different solvents extract. The highest phenolic contents (137, 136 and 135 μg GAE/mg extract) was found in Met + WT, Acet and Met+Eth extracts respectively.

FIGURE 3
Total phenolic content μg/mg extract gallic Acid equivalent (GAE).

HPLC-DAD fingerprinting

High performance liquid chromatography-Diode Array detection (HPLC-DAD) analysis of methanolic, chloroform, ethylacetate and n-hexane fractions was performed (Figure 4 A, B, C, D). In case of methanolic fraction, highest concentrated signals were observed at retention times of 33.1 (Quercetin-3-O-glucoside), 34.0 (Rosmarinic acid), 31.0 (HHDP-galloyl-glucose), 27.8 (Chicoric acid) min, which were 58.6, 15.0, 7.8 and 6.47 % respectively. In case of chloroform fraction, highest concentrated signals were observed at retention times of 32.9 (Quercetin), 33.7 (Phloroglucinol), 30.9 (Quercetin), 29.6 (Caffeic acid derivative) min, which were 46.8, 14.2, 8.8 and 8.2 % respectively. In case of ethylacetate fraction, highest concentrated signals were observed at retention times of 33.1 (Quercetin-3-O-glucoside), 34.0 (Rosmarinic acid), 31.1 (Quercetin-3-(caffeoyldiglucoside)-7-glucoside), 26.9 (p-Coumaric acid) min, which were 58.4, 16.7, 8.3 and 5.8 % respectively while for n-hexane, highest concentrated signals were observed at retention times of 33.1 (Quercetin-3-O-glucoside), 30.1 (Mandalic acid), 22.8 (Proanthocyanidin B1), 31.0 (HHDP-galloyl-glucose) min, which were 38.4, 13.3, 12.5 and 7.4 % respectively. The compounds were identified by comparing absorption spectra of the sample with the standard compounds or from the values reported in the literature and our previously published papers (Aaby, Ekeberg, Skrede 2007Aaby K, Ekeberg D, Skrede G. Characterization of phenolic compounds in strawberry (Fragaria× ananassa) fruits by different HPLC detectors and contribution of individual compounds to total antioxidant capacity. J Agric Food Chem. 2007;55(11):4395-4406.; Mradu et al., 2012Mradu G, Saumyakanti S, Sohini M, Arup M. HPLC profiles of standard phenolic compounds present in medicinal plants. Intern. J Pharmacog Phytochem Res. 2012;4(3):162-167.; Ibrahim et al., 2015Ibrahim RM, El-Halawany AM, Saleh DO, El Naggar EMB, El-Shabrawy AE-RO, El-Hawary SS. HPLC-DAD-MS/MS profiling of phenolics from Securigera securidaca flowers and its anti-hyperglycemic and anti-hyperlipidemic activities. Rev Bras Farmacogn. 2015;25(2):134-141.; Ovais et al., 2018bOvais M, Ayaz M, Khalil AT, Shah SA, Jan MS, Raza A, et al. HPLC-DAD finger printing, antioxidant, cholinesterase, and α-glucosidase inhibitory potentials of a novel plant Olax nana. BMC Compl Alternative Med . 2018b;18(1):1.). The current pharmacological effects of solvent extracts can be attributed to the presence of these compounds.

FIGURE 4
HPLC-DAD finger printing of A) methanolic fraction, B) chloroform fraction C) Ethylacetate fraction and D) n-Hexane fraction.

Antioxidant Assays

Total antioxidant capacity (Phosphomolybdenum method)

Figure 5 shows the total antioxidant capacity of different solvent extracts. Among all extracts, highest TAC was shown by Met+Eth extract (392μg AAE /mg extract) followed by Acet extract (301 μg AAE /mg extrac) and Met (275 μg AAE /mg extract). The lowest antioxidant activity was shown by Eacet (63 μg AAE / mg extract) and NH (17 μg AAE /mg extract).

FIGURE 5
Total antioxidant capacity (μg/mg extract ascorbic acid equivalent (AAE) of different solvent extracts of E. intermedia.

DPPH free radical scavenging

In case of DPPH free radical scavenging, maximum scavenging potential was observed in Met+Eth extract (74.9%) followed by Acet (74.5%) and Met (74%) extracts as shown in Figure 6. The minimum activity was shown by NH extract (10%).

FIGURE 6
Percent free radical scavenging activity of different solvent extracts of E. intermedia.

Reducing power

The highest reducing power was detected in Eth extract (271 μg AAE/mg extract) followed by Acet extract (236 μg AAE/mg extract) as shown in Figure 7.

FIGURE 7
Total reducing power of different solvent extracts of E. intermedia.

Antibacterial activity

The antibacterial potential of each solvent extracts of E. intermedia is shown in Table II. Growth inhibitory zone was calculated through agar well diffusion method and MIC was determined by broth micro dilution method. Among all solvents extract, Eth+Met extract was found effective against B. subtilis, K. pneumoniae and S. epidermidis, producing zones of inhibition of 15 ± 0.04, 15 ± 0.72 and 14 ± 0.33 mm with MIC of 11.1, 11.1 and 33.3 μg/mL respectively. Eth extract was found active against B. subtilis, E.coli, P. aeruginosa and K. pneumonia. Met extract showed significant activity against (MIC 33.3 μg/mL) against P. aeruginosa.

TABLE II
Antibacterial activities of different solvent extract of E. intermedia

Antifungal activity

In case of antifungal assay, the highest zone of inhibition (15 ± 0.31 mm) was shown by Eth extract against A. fumigatus followed by Met + WT extract (14 ± 0.66 mm) against A. niger shown in Table III. Eth + Met extract was found to be active against Mucor spp. only and Met+WT gave highest zone of inhibition (11 ± 0.69 mm) among all extracts against A. flavus.

TABLE III
Antifungal activities of different solvents extracts of E. intermedia

Percent hemolysis

The lowest % hemolysis was shown by Eth extract (1.98%) followed by Met, Acet and Met + Eth with % hemolysis of 2, 3 and 3% respectively as shown in Figure 8. The highest % hemolysis was shown by NH and Chlo extracts i.e. 43.56 and 30.81% respectively.

FIGURE 8
Effect of different solvent extracts of E. intermedia on human erythrocytes.

Anti-tumoral potential

Each solvent extracts were appraised for their anticancer potential against HepG2 liver cancer cell line as shown in Figure 8. Among all extracts, Met + WT, Eth + Met and Eth extract exhibited significant anticancer activities with IC 50 values of 13.51, 16.12 and 19.23 μg/ mL respectively while standard drug vincristine showed IC50 of 7.3 μg/mL (Figure 9).

FIGURE 9
Cytotoxic effects and IC50 of different solvent extracts of E. intermedia on HepG2 liver cancer cell lines.

DISCUSSION

As the extraction procedure involves the solubility of some phytochemicals on polarity basis, it is significant to investigate the correlation between the extraction method applied and their physiochemical properties (Tabassum et al., 2017Tabassum S, Ahmed M, Mirza B, Naeem M, Zia M, Shanwari ZK, et al. Appraisal of phytochemical and in vitro biological attributes of an unexplored folklore: Rhus Punjabensis Stewart. BMC Compl Alternative Med . 2017;17(1):146.). Chloroform and Eacet extract were found to have high flavonid content. Different flavonoids such as C-glycosyl flavones, flavonol-3-O-glycosides and proanthocyanidins have been reported for different species of Ephedra (Wallace et al. 1982Wallace JW, Porter PL, Besson E, Chopin J. C-glycosylflavones of the gnetopsida. Phytochemistry . 1982;21(2):482-483.; Boulos, 1983Boulos L. Medicinal plants of North Africa. Medicinal plants of North Africa. 1983.; Nawwar, El-Sissi HI, Barakat, 1984Nawwar MA, El-Sissi HI, Barakat HH. Flavonoid constituents of Ephedra alata. Phytochemistry . 1984;23(12):2937-2939.; Hussein et al., 1997Hussein SA, Barakat HH, Nawar MA, Willuhn G. Flavonoids from Ephedra aphylla. Phytochemistry. 1997;45(7):1529-1532.).

Both edible and non-edible plants contains different phenolic compounds (Kumaran, Karunakaran, 2007Kumaran A, Karunakaran RJ. In vitro antioxidant activities of methanol extracts of five Phyllanthus species from India. LWT-Food Sci Technol. 2007;40(2):344-352.). As phenolic compounds are very responsive towards free radicals, so the quantification of these compounds correlate to antioxidant activity (Loizzo et al., 2012Loizzo MR, Tundis R, Bonesi M, Menichini F, Mastellone V, Avallone L, et al. Radical scavenging, antioxidant and metal chelating activities of Annona cherimola Mill.(cherimoya) peel and pulp in relation to their total phenolic and total flavonoid contents. J Food Compost Anal. 2012;25(2):179-184.). The genus Ephedra is reported to contain different phenolic compounds (Parsaeimehr, Sargsyan, Javidnia, 2010Parsaeimehr A, Sargsyan E, Javidnia K. A comparative study of the antibacterial, antifungal and antioxidant activity and total content of phenolic compounds of cell cultures and wild plants of three endemic species of Ephedra. Molecules. 2010;15(3):1668-1678.; Jaradat, Hussen, Al Ali, 2015Jaradat N, Hussen F, Al Ali A. Preliminary phytochemical screening, quantitative estimation of total flavonoids, total phenols and antioxidant activity of Ephedra alata Decne. J Mater Environ Sci. 2015;6(6):1771-1778.). It is reported that methanolic extract of E. procera showed high phenolic contents (Parsaeimehr, Sargsyan, Javidnia, 2010Parsaeimehr A, Sargsyan E, Javidnia K. A comparative study of the antibacterial, antifungal and antioxidant activity and total content of phenolic compounds of cell cultures and wild plants of three endemic species of Ephedra. Molecules. 2010;15(3):1668-1678.). Nebrodenside A and nebrodenside B are phenolic glycosides isolated from the aerial parts of E. nebrodensis (Cottiglia et al., 2005Cottiglia F, Bonsignore L, Casu L, Deidda D, Pompei R, Casu M, et al. Phenolic constituents from Ephedra nebrodensis. Nat Prod Res . 2005;19(2):117-123.). Different species of genus Ephedra such as E. major, E. distachya subsp. helvetica, E. fragilis E. major, E. distachya sub sp. E. fragilis, E. foeminea, E. altissima, E. alata, and E. foliate have already been evaluated for their flavonoids and phenolic contents (Ibragic, Sofić, 2015Ibragic S, Sofić E. Chemical composition of various Ephedra species. Bosn. J Basic Med Sci. 2015;15(3):21.; Aghdasi et al., 2016Aghdasi M, Mofid Bojnoordi M, Mianabadi M, Nadaf M. Chemical components of the Ephedra major from Iran. Nat Prod Res. 2016;30(3):369-371.). In HPLC-DAD analysis, in case of methanolic fraction, highest concentrated signals were observed at retention times of 33.1 (Quercetin-3-O-glucoside), 34.0 (Rosmarinic acid), 31.0 (Galloyl-Hexahydroxydiphenoyl-galloyl-glucose), 27.8 (Chicoric acid) min, which were 58.6, 15.0, 7.8 and 6.47 % respectively. In case of chloroform fraction, highest concentrated signals were observed at retention times of 32.9 (Quercetin), 33.7 (Phloroglucinol), 30.9 (Quercetin), 29.6 (Caffeic acid derivative) min, which were 46.8, 14.2, 8.8 and 8.2 % respectively. In case of ethylacetate fraction, highest concentrated signals were observed at retention times of 33.1 (Quercetin-3-O-glucoside), 34.0 (Rosmarinic acid), 31.1 (Quercetin-3-(caffeoyldiglucoside)-7-glucoside), 26.9 (p-Coumaric acid) min, which were 58.4, 16.7, 8.3 and 5.8 % respectively while for n-hexane, highest concentrated signals were observed at retention times of 33.1 (Quercetin-3-O-glucoside), 30.1 (Mandalic acid), 22.8 (Proanthocyanidin B1), 31.0 (Galloyl-Hexahydroxydiphenoyl-galloyl-glucose) min, which were 38.4, 13.3, 12.5 and 7.4 % respectively.

Among all different solvent extracts, the highest and lowest total antioxidant capacity was observed in Met+Eth and NH extracts respectively. Phenolic compounds such as gallic acid, rutin and catechin are responsible for antioxidant activity as these compounds donate hydrogen atom or chelate metals which results in interruption of chain oxidation reactions. The high amounts of phenolic compounds are responsible for high antioxidant activity (Viuda-Martos et al., 2010Viuda-Martos M, Ruiz Navajas Y, Sánchez Zapata E, Fernández-López J, Pérez-Álvarez JA. Antioxidant activity of essential oils of five spice plants widely used in a Mediterranean diet. Flavour Fragr J. 2010;25(1):13-19.).

The ability of antioxidants present in extracts to decolorize 2, 2-diphenyl-2-picryl-hydrazyl (DPPH) is the basic principle of free radical scavenging assay. Highest free radical scavenging potential was observed in Met+Eth extract while lowest free radical scavenging potential was observed in NH extract. The results of free radical scavenging potential and total phenolic contents were found to have positive correlation. It is reported that phenolic compounds possess free radical scavenging properties (Tabassum et al., 2017Tabassum S, Ahmed M, Mirza B, Naeem M, Zia M, Shanwari ZK, et al. Appraisal of phytochemical and in vitro biological attributes of an unexplored folklore: Rhus Punjabensis Stewart. BMC Compl Alternative Med . 2017;17(1):146.). E. nebrodensis has been also reported for its antioxidant potential (Ballero et al., 2010Ballero M, Foddis C, Sanna C, Scartezzini P, Poli F, Petitto V, et al. Pharmacological activities on Ephedra nebrodensis Tineo. Nat Prod Res . 2010;24(12):1115-1124.).

Reducing power of the extract is one of the prominent indicator that the extract possesses antioxidant potential. The Eth extract was found to have maximum reducing power. The results show that total phenolic content and reducing potential have positive correlation. Ephedrine and pseudoephedrine are its two primary active ingredients, which possess a variety of biological activities. However, the active components are still unknown and future research is required to isolate and characterize compounds responsible for their biological activities (Mir et al., 2019Mir NT, Saleem U, Anwar F, Ahmad B, Ullah I, Hira S, et al. Lawsonia Inermis markedly improves cognitive functions in animal models and modulate oxidative stress markers in the brain. Medicina. 2019;55(5):192.; Saleem et al., 2021Saleem U, Akhtar R, Anwar F, Shah MA, Chaudary Z, Ayaz M, et al. Neuroprotective potential of Malva neglecta is mediated via down-regulation of cholinesterase and modulation of oxidative stress markers. Metabolic Brain Dis. 2021;1-12.).

Antibiotic resistance is becoming global issue and there is urgent need to search novel antibacterial agents (Ovais et al., 2018cOvais M, Khalil AT, Raza A, Islam NU, Ayaz M, Saravanan M, et al. Multifunctional theranostic applications of biocompatible green-synthesized colloidal nanoparticles. Appl Microbiol Biotechnol . 2018c;102(10):4393-4408.; Ovais et al., 2019Ovais M, Zia N, Khalil AT, Ayaz M, Khalil A, Ahmad I. Nanoantibiotics: recent developments and future prospects. frontiers in clinical drug research-anti infectives: Volume 5. 2019;5158.). Plants are one of the potential source of innovative antibacterial agents as they contain many bioactive compounds (Bissa, 2015Bissa S. Evaluation of antibacterial potential of Ephedra foliata Boiss Ex CA Mey. Bioscan. 2015;10(3):1169-1172.; Ayaz, et al. 2017dAyaz M, Subhan F, Sadiq A, Ullah F, Ahmed J, Sewell R. Cellular efflux transporters and the potential role of natural products in combating efflux mediated drug resistance. Front Biosci. (Landmark edition). 2017d;22:732-756.). Chlo extract showed activity against S. epidermidis only while S. aureus strain was found resistant against all solvent extracts. The significant antibacterial activities of Eth + Met, Eth and Met extracts against B. subtilis, K. pneumonia, S. epidermidis, P. aeruginosa and E.coli may be due to phenolic compounds like protocatechuic acids, caffeic acid, p-hydroxybenzoic, p-coumaric acids, oleuropein and vanillic present in the extracts (Aziz et al., 1998Aziz N, Farag S, Mousa L, Abo-Zaid M. Comparative antibacterial and antifungal effects of some phenolic compounds. Microbios. 1998;93(374):43-54.). The antimicrobial potential of medicinal plants extract is mostly based upon solvent used for extraction, tested organism and part of the plant used (Tabassum et al., 2017Tabassum S, Ahmed M, Mirza B, Naeem M, Zia M, Shanwari ZK, et al. Appraisal of phytochemical and in vitro biological attributes of an unexplored folklore: Rhus Punjabensis Stewart. BMC Compl Alternative Med . 2017;17(1):146.). Different species of Ephedra have been already evaluated for the antibacterial potential (Dashtdar, Dashtdar, Dashtdar, 2013Dashtdar M, Dashtdar MR, Dashtdar B. In-Vitro, Anti-Bacterial Activities of Aqueous Extracts of Acacia catechu (LF) Willd, Castanea sativa, Ephedra sinica stapf and Shilajita mumiyo Against Gram Positive and Gram Negative Bacteria. J Pharmacopunct. 2013;16(2):15.; Khan et al., 2017Khan A, Jan G, Khan A, Gul Jan F, Bahadur A, Danish M. In Vitro Antioxidant and Antimicrobial Activities of Ephedra gerardiana (Root and Stem) Crude Extract and Fractions. Evid-based Compl Alt Med. 2017;2017.) Castanea sativa, Ephedra sinica stapf and shilajita mumiyo Against Gram Positive and Gram Negative Bacteria In Vitro Antioxidant and Antimicrobial Activities of Ephedra gerardiana (Root and Stem. Methanol extract of E. sarcocarpa growing in Iran showed good activity against E. coli and P. aeruginosa (Rustaiyan et al. 2011Rustaiyan A, Javidnia K, Farjam MH, Aboee-Mehrizi F, Ezzatzadeh E. Antimicrobial and antioxidant activity of the Ephedra sarcocarpa growing in Iran. J Med Plants Res. 2011;5(17):4251-4255.). Petroleum ether extract of Ephedra foliate stem and leaves showed maximum activity against Klebsiella pneumoniae and Enterobacter aerogenes respectively (Bissa, 2015Bissa S. Evaluation of antibacterial potential of Ephedra foliata Boiss Ex CA Mey. Bioscan. 2015;10(3):1169-1172.). Methanol extract of Ephedra alataalenda has high antibacterial activity against Enterococcus facealis and Baccillus subtilus (Jerbi et al., 2016Jerbi A, Zehri S, Abdnnabi R, Gharsallah N, Kammoun M. Essential Oil Composition, Free-Radical-Scavenging and Antibacterial Effect from Stems of Ephedra alata alenda in Tunisia. J Essent Oil-Bear Plants. 2016;19(6):1503-1509.)++++++.

The tested fungal strains (A. niger, A. fumigatus, Mucor species and A. flavus) have been reported to cause different human diseases (Waxman et al., 1987Waxman JE, Spector JG, Sale SR, Katzenstein ALA. Allergic Aspergillus sinusitis: concepts in diagnosis and treatment of a new clinical entity. Laryngoscope. 1987;97(3):261-266.; Gugnani et al., 1989Gugnani H, Okafor B, Nzelibe F, Njoku-Obi A. Etiological Agents of Otomycosis in Nigeria Das Erregerspektrum von Otomykosen in Nigeria. Mycoses. 1989;32(5):224-229.; Pai, Platt, 1995Pai S, Platt M. Antifungal effects of Allium sativum (garlic) extract against the Aspergillus species involved in otomycosis. Lett Appl Microbiol. 1995;20(1):14-18.). In our study, Eth extract showed high antifungal activity against A. fumigatus while Met + WT extract showed high activity against A. niger. Our results can be justified by previous studies (Bonjar, 2004Bonjar GS. Anti yeast activity of some plants used in traditional herbal medicine of Iran. J Biol Sci. 2004;4(212):5.; Parsaeimehr, Sargsyan, Javidnia, 2010Parsaeimehr A, Sargsyan E, Javidnia K. A comparative study of the antibacterial, antifungal and antioxidant activity and total content of phenolic compounds of cell cultures and wild plants of three endemic species of Ephedra. Molecules. 2010;15(3):1668-1678.)antifungal and antioxidant activity and total content of phenolic compounds of cell cultures and wild plants of three endemic species of Ephedra. Other species of genus Ephedra such as E. major Host (Bagheri-Gavkosh et al., 2009Bagheri-Gavkosh S, Bigdeli M, Shams-Ghahfarokhi M, Razzaghi-Abyaneh M. Inhibitory effects of Ephedra major host on Aspergillus parasiticus growth and aflatoxin production. Mycopathologia. 2009;168(5):249.), E. sinica (Wanlong, Lizhen, 1995Wanlong ZGFYD, Lizhen X. A study on antifungal activity of essential oils from Ephedra sinica and asarum heterotropoides var. Mandshuricum [J]. J Plant Prot. 1995;4014.) and Ephedra foliata Bois (Mewari, Kumar, 2011Mewari N, Kumar P. Evaluation of antifungal potential of Marchantia polymorpha L., Dryopteris filix-mas (L.) Schott and Ephedra foliata Boiss. against phyto fungal pathogens. Arch Phytopathol Plant Protec. 2011;44(8):804-812.) have been reported for their antifungal potential.

The in vitro cytotoxity on the red cells was evaluated by using different solvent extracts. There are many phytochemicals in medicinal plants which can disrupt the biological membranes therefore it is necessary to check the effect of extract on red blood cells (Sharma, Sharma, 2001Sharma P, Sharma JD. In vitro hemolysis of human erythrocytes-by plant extracts with antiplasmodial activity. J Ethnopharmacol. 2001;74(3):239-243.). Evaluation of phytotherapeutic products must be routinely considered before the product is marketed as the use of these products is increasing globally and can cause serious public health problems (Secco, 1990Secco RdS. Produtos naturais: alternativa segura? Ciênc Cult. (São Paulo). 1990;42(10/12):807-810.; Willcox, Ash, Catignani, 2004Willcox JK, Ash SL, Catignani GL. Antioxidants and prevention of chronic disease. Crit Rev Food Sci Nutr. 2004;44(4):275-295.; de Freitas et al., 2008de Freitas MV, Rita de Cássia MN, da Costa Huss JC, de Souza TMT, Costa JO, Firmino CB, et al. Influence of aqueous crude extracts of medicinal plants on the osmotic stability of human erythrocytes. Toxicology in Vitro. 2008;22(1):219-224.). RBCs provide a good platform and experimental sample for the study of membrane stability. After lysis of RBCs, hemoglobin is released and quantified through spectrophotometer. In our study, Eth and Met extract showed minimum % hemolysis while maximum % hemolysis was shown by NH and Chlo extracts. Our results are supported by previous work (Erdem et al., 2013Erdem AK, Aydoğdu EA, Öz GC, Erol Ç, Yazgan M. Antibacterial and hemolytic activities of different extracts of Amsonia orientalis Decne (Apocynaceae). IUFS J Biol. 2013;72(1):1-8.),”even_P–A, which reported that chloroform extract possess high hemolytic activity.

In our study, it was found that Met + WT and Eth + Met extracts showed high anticancer potentials. The anticancer potential might be due to the active compounds present in the extract that are responsible for changing the signal transduction pathways; kinase inhibitor, involved in cell cycle arrest (Ovais et al., 2017Ovais M, Raza A, Naz S, Islam NU, Khalil AT, Ali S, et al. Current state and prospects of the phytosynthesized colloidal gold nanoparticles and their applications in cancer theranostics. Appl Microbiol Biotechnol . 2017;101(9):3551-3565.; Ovais et al., 2018c; Ayaz et al., 2019bAyaz M, Ullah F, Sadiq A, Ullah F, Ovais M, Ahmed J, et al. Synergistic interactions of phytochemicals with antimicrobial agents: Potential strategy to counteract drug resistance. Chem Bio. Interac. 2019b;308:294-303), inhibitory effect of flavonoids on protein kinases, different transcriptional factors (Miranda et al., 1999Miranda C, Stevens J, Helmrich A, Henderson M, Rodriguez R, Yang Y-H, et al. Antiproliferative and cytotoxic effects of prenylated flavonoids from hops (Humulus lupulus) in human cancer cell lines. Food Chem Toxicol. 1999;37(4):271-285.) and extract may contain molecules which inhibit the interaction of (MDM2) murine double minute and p53. MDM2 is mostly over expressed in cancerous cells and it results in inactivation of p53 (Riaz et al., 2017Riaz M, Ashfaq UA, Qasim M, Yasmeen E, Qamar MTU, Anwar F. Screening of medicinal plant phytochemicals as natural antagonists of p53-MDM2 interaction to reactivate p53 functioning. Anticancer Drugs. 2017;28(9):1032-1038.).

CONCLUSION

In present study, different solvent extracts of E. intermedia were evaluated for their phytochemical analysis and in vitro biological activities. We have reported the use of different solvent system for the comprehensive phytochemical as well as biological analysis of subjected plant. The results showed that the diverse biological activities of extracts depend on the organic solvent in which the extraction is done. Polar solvent extracts may contain phytochemicals responsible for good antioxidant potentials as compared to non-polar solvent extracts. This plant can be used for isolation of antioxidants, antimicrobial agents and anticancer compounds. Moreover, all the reported activities need isolation of bioactive compounds through HPLC, GCMS and NMR based on the reported results of biological activities.

ACKNOWLEDGEMENTS

MoSAEL Lab, Department of Biotechnology, Quaid-i-Azam University, Islamabad is highly acknowledged for providing laboratory facilities to conduct experimental work.

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DECLARATIONS

  • Approval of Ethical Committee

    The study was approved by Research Ethics Committee, Department of Biotechnology, Quaid-i-Azam University Islamabad, Pakistan, Dated 15-1-2017 via reference No: DREC/BIO/20170600/07.
  • Consent to publish

    Not applicable in this section
  • Availability of data and materials

    The data presented in this manuscript belong to research work of Muhammad Qasim Nasar and has not been deposited in any repository yet. However, the materials are available to the researchers upon request.
  • Funding

    This research has received no specific grant from any funding agency in the public, commercial, or not for-profit sectors.

Publication Dates

  • Publication in this collection
    23 Jan 2023
  • Date of issue
    2022

History

  • Received
    27 Oct 2020
  • Accepted
    20 Feb 2021
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