Journal of Traditional and Complementary Medicine 10 (2020) 13e25
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Journal of Traditional and Complementary Medicine
journal homepage: http://www.elsevier.com/locate/jtcme
Lycium shawii Roem. & Schult.: A new bioactive antimicrobial and antioxidant agent to combat multi-drug/pan-drug resistant pathogens of wound burn infections
* Sameh S. Ali a, b, , Nessma A. El-Zawawy b, Rania Al-Tohamy a, Shimaa El-Sapagh b, ** Ahmed M. Mustafa c, d, Jianzhong Sun a, a Biofuels Institute, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, China b Botany Department, Faculty of Science, Tanta University, Tanta 31527, Egypt c College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China d Department of Agricultural Engineering, Faculty of Agriculture, Suez Canal University, Ismailia 41522, Egypt article info abstract
Article history: The Multidrug Drug Resistance (MDR) and Pan-Drug Resistance (PDR) remain an intractable challenge Received 12 October 2018 issue in public health, worldwide. Plant extracts-based biological macromolecules containing a diverse Received in revised form array of secondary metabolites could be potentially used as alternative approaches to control or limit 3 January 2019 MDR/PDR infections. Plants of the Solanaceae family exhibit a wide variety of secondary metabolites with Accepted 8 January 2019 antioxidant and antimicrobial properties, which render them a significant role in food and pharma- Available online 12 January 2019 ceutical applications. To our knowledge, this is the first report on phytochemical constituents, antioxi- dant, antimicrobial activities and in vivo toxicological safety of Lycium shawii leaf extracts. Results Keywords: revealed that phenolics and flavonoids were found to be the most abundant compounds in all extracts. Pseudomonas aeruginosa þ Staphylococcus aureus Antioxidant activity of extracts was measured using DPPH and ABTS assays and the methanol extract þ Aspergillus niger displayed superior scavenging activity (IC50 ¼ 0.06 and 0.007 mg/mL for DPPH and ABTS , respectively). Candida albicans Results of the GC-MS analysis revealed the identity of 10 compounds. Moreover, in vivo toxicological Pharmaceutical applications assessment can confirm the safety of L. shawii for use. Overall, L. shawii leaves are a promising natural Herbal medicine source for the development of novel antimicrobial and antioxidant agents that could potentially combat clinical MDR/PDR pathogens. © 2019 Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
1. Introduction classes is defined as PDR.1 Several drug-resistant strains including Pseudomonas aeruginosa (Gram-negative bacteria), Staphylococcus In recent years, nosocomial infections by bacterial and fungal aureus (Gram-positive bacteria), Aspergillus niger (filamentous pathogens conferring Multi-Drug Resistance (MDR) or Pan-Drug fungi) and Candida albicans (unicellular fungi) have been known as e Resistance (PDR) to various commercially available antibacterial most terrible pathogens of burn infections,1 8 to which effective and antifungal agents have become an intractable challenging issue alternative remedies are urgently required. Therefore, it is crucial to worldwide. MDR is defined as resistance to three or more antimi- discover novel antimicrobial agents that can potentially in demand crobial classes. However, resistance to all agents in all antimicrobial to combating drug resistance, as well as to treat these life- threatening infections.9,10 Till date, biological properties and bioactive compounds of many medicinal plants are not studied.11 Traditionally used medicinal * Corresponding author. Biofuels Institute, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, China. plants produce a diverse array of bioactive phytoconstituents such ** Corresponding author. as phenolics, alkaloids, flavonoids etc. These phytochemicals or E-mail addresses: [email protected], [email protected] (S.S. Ali), phytobiotics derived from plant sources in the form of extracts [email protected] (J. Sun). displayed superiority over chemical approaches, and they have Peer review under responsibility of The Center for Food and Biomolecules, shown the potential to avert the danger caused by deadly National Taiwan University. https://doi.org/10.1016/j.jtcme.2019.01.004 2225-4110/© 2019 Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 14 S.S. Ali et al. / Journal of Traditional and Complementary Medicine 10 (2020) 13e25
Abbreviations MBC Minimum Bactericidal Concentration MDR Multi-Drug Resistance ABTS 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonic MFC Minimum Fungicidal Concentration acid) MIC Minimum Inhibitory Concentration
AlCl3 Aluminum trichloride NIST National Institute of Standard and Technology DMSO Dimethyl sulfoxide OECD Organization for Economic Cooperation and DIZ Diameter Inhibition Zone Development DPPH 1,1-diphenyl-2-picrylhydrazyl PBP Penicillin-Binding Protein GAE Gallic Acid Equivalent PDR Pan-Drug Resistance GC-MS Gas Chromatography-Mass Spectrum QE Quercetin GSH Glutathione TEM Transmission Electron Microscopy HPLC High Performance Liquid Chromatography TFC Total Flavonoid Content IAEC Institutional Animal Ethics Committee TPC Total Phenolic Content
LD50 Lethal Dose 50
e infections.12 14 Hamidpour et al.15 reported that the Russian olive safety using acute oral toxicity and haemolytic activity for (Elaeagnus angustifolia L.) extract has novel roles as active antioxi- completeness of its assessment as a new leading structure in the dant, anti-inflammatory and analgesic agent. This extract has pharmaceutical field. shown to be effective in reducing the healing time of wounds in injured patients. The leaf extract of Alpinia nigra possesses pro- 2. Materials and methods found antibacterial, analgesic and cytotoxic activities.14 Heidari et al.13 reported the efficacy of topical application of a standardized 2.1. Plant collection extract of Tragopogon graminifolius in the healing process of burn wounds. They concluded that this extract could be considered as a Egyptian Lycium shawii Roem. & Schult. leaves were collected future treatment for healing of burn wounds. Many commercially from El-Omayed Biosphere Reserve area at the Northern Mediter- established antibiotics incorporated in the present-day use medi- ranean Coast of Egypt (Supplemental information, Fig. S1). Table 1 cine has been suggested potentially useful biological activity.16 depicts the botanical name, Arabic name, family, habit, habitat, Interestingly, the principle advantages of employing plant-based distribution and medical uses of L. shawii.16 The leaves were antimicrobials are that they are relatively safer and affordable washed with tap water, dried and pulverized to a fine powder by e than synthetic alternatives.17 21 using a lab grinder. The resultant powder was stored for further Wang et al.22 have proposed several mechanisms of antibacte- experiments. rial and antifungal potentials of ethnomedicinal plants, which include decomposition of the cytoplasmic membrane, disturbance 2.2. Crude extraction of outer membrane of Gram-negative bacteria, accretion of ATPase, inhibition of enzymes synthesis, and coagulation of cell inner A total of 50 g of dried L. shawii leaves was extracted with contents. It is reported that Gram-negative bacteria are more methanol using Soxhlet apparatus for 5e7 h at 50 C. The solvent resistant towards the antibacterial activities of medicinal plant used for extraction was evaporated using rotary evaporator under extracts as compared to Gram-positive bacteria and this is attrib- reduced pressure. The crud extract slurry formed was thoroughly uted to the presence of outer membrane structure in Gram- dried and weighted. Ethanol, ethyl acetate and aqueous (water) negative cells that reduced the penetration rate of compounds extracts were similarly performed. All extracts were freeze-dried with antimicrobial activities.9,10 On the other hand, the mecha- at 20 C and stored at 4 C until use. nisms of antifungal actions are consonant to those described above for bacteria, including irreversible trauma to the cell septum, 2.3. Phytochemical screening exuding and coagulation of cellular interior materials.23 Plants of the Solanaceae family exhibit a wide variety of sec- Phytochemical components (flavonoids, alkaloids, phenolics, ondary metabolites with different biological activities including tannins, saponins, steroids, coumarins, glycosides and terpenoids) nutritional antioxidants, antimicrobials, anti-inflammatory, anti- were screened in the four extracts of L. shawii leaves using standard cancer or cytotoxic properties, which render them to play signifi- conventional protocols.28 e cant roles in food and pharmaceutical applications.24 26 Recent phytochemical studies confirmed that the richness of numerous 2.4. Quantitative assay of total bioactive components constituents, such as terpenoids, alkaloids and flavonoids endows Lycium species (Solanaceae family) with a variety of biological ac- Phenolics and flavonoids were found to be the most abundant 16 tivities. The fruits, flowers and roots of many Lycium species have compounds in L. shawii extracts. Therefore, Total Flavonoid Content 27 long been used as an important source of food and/or medicine. (TFC) and Total Phenolic Content (TPC) were determined as However, to the best of the authors' knowledge, to date, there described below. have been no reports to investigate the leaf extract of Lycium shawii as an alternative source of natural antioxidant and antimicrobial 2.4.1. Total Flavonoid Content (TFC) agent combating MDR/PDR pathogens of burn wound infections. A total of 500 mL sample solution was mixed with the same Therefore, this study was aimed at evaluating the novel pharma- volume of aluminum trichloride (AlCl3; 2%) in methanol. A blank ceutical potentials of the traditional medicinal plant, L. shawii,as was prepared by adding a sample solution to methanol without active antimicrobial against MDR/PDR clinical pathogens of burn AlCl3. Similarly, the procedures were repeated for other L. shawii wound infections, antioxidant agent and with its toxicological extracts. After a 10 min incubation at room temperature, blank and S.S. Ali et al. / Journal of Traditional and Complementary Medicine 10 (2020) 13e25 15
Table 1 Ethnobotanical value of Lycium shawii used in this study.
Botanical Arabic Family Habit Habitat Distribution Medicinal uses Other uses Reference name name
Lycium Awsaj Solanaceae Shrub Sandy and silty depressions, Recorded in Egypt, Qatar, A diuretic, Grazed by animals and the berries are 16 shawii runnels, wadis and on rocky Bahrain, Kuwait, Saudi laxative and eaten by man; the leaves provide Roem. & slopes Arabia and UAE tonic agent browsing for camels Schult.
sample solutions were measured at 415 nm using a UVevisible results, four strains; namely P. aeruginosa 9 (PA-09), S. aureus 17 spectrophotometer (Shimadzu-UV2600, Japan). TFC was (SA-17), A. niger 5 (AN-05) and C. albicans 11 (CA-11) were selected expressed as milligrams of quercetin (QE) equivalents (mg QE/g for further experiments. L. shawii extract was dissolved in pure extract). dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, Missouri, USA) to a final concentration of 100 mg plant extract in 1 mL of DMSO. 2.4.2. Total Phenolic Content (TPC) The extract was sterilized by filtration on 0.45 mm Millipore filters. A total of 250 mL sample solution was mixed with 1000 mL Then, 20 mL of this extract was soaked into sterile filter paper discs. FolineCiocalteu reagent (1:9; v/v) and the mixture was incubated These discs were placed on Muller-Hinton agar plates previously m for 3 min. Then, the Na2CO3 solution (750 mL; 1%) was added. After a swabbed with 100 L of bacterial and fungal inoculum (approxi- 2 h incubation at room temperature, the sample absorbance was mately 108 CFU/mL). The DMSO was used as a negative control. The measured at 760 nm using a UVevisible spectrophotometer (Shi- MIC and MFC of L. shawii leaf extracts were performed by a serial madzu-UV2600, Japan) with a constructed calibration curve using dilution technique.31 MIC and MFC were regarded as the lowest gallic acid. TPC was expressed as milligrams of Gallic Acid Equiva- concentration L. shawii extract at which no change the color lents (mg GAE/g extract). occurred.
2.5. Antioxidant activity 2.8. Transmission Electron Microscopy (TEM)
Phenolic and flavonoid compounds are associated with antiox- The mechanism of antimicrobial action of the L. shawii methanol idant activity. Therefore, it is important to assess the antioxidant extract was performed using TEM as we previously described in activity of L. shawii leaf extracts, and this assessment was per- details.3,6,32 Briefly, DMSO-treated cells (control), cells incubated formed as described below. without DMSO (control), and cells treated with the MIC of L. shawii extract were prepared. Samples were examined using a TEM (JEOL- 2.5.1. 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH ) assay JEM-100SX, Japan). The scavenging activity against DPPH was estimated.29 The assay depends on the reduction of DPPH (purple color) to a 2.9. Gas Chromatography-Mass Spectrum (GC-MS) analysis diphenyl picrylhydrazine (yellow color) and the remaining DPPH was measured at 517 nm after a 30 min incubation at 37 C in the The methanol extract of L. shawii was concentrated in a desic- dark. Glutathione (GSH) was used as a positive control. The DPPH cator and subjected to GC-MS analysis. GC-MS model Agilent scavenging activity was expressed as milligrams of GSH equivalents 8790B/5977B MSD was used in the analysis that employs Agilent (mg GSH/g extract). 19091s-433UI Hp-5 column (60e325 C) and the components were separated using helium (He) as a carrier gas at a constant flow of 2.5.2. 2,20-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) 0.8 mL/min at ionization energy (70eV). The sample extract (1 mL) þ radical cation (ABTS ) assay was injected into the instrument. The injector temperature was set þ The scavenging activity against ABTS was estimated.29 The at 250 C and throughout the analysis, temperature flow was set at sample absorbance was measured at 734 nm after a 30 min incu- the speed of increasing 10 C/min. The name, molecular weight, bation at 37 C. The 6-hydroxy-2,5,7,8-tetramethylchromane-2- compound nature and a molecular formula of the test sample were carboxylic acid (Trolox; Sigma, USA) was used as a positive con- identified based on the National Institute of Standard and Tech- þ trol. The ABTS scavenging activity was expressed as milligrams of nology (NIST) library spectra data bases. Trolox equivalents (mg Trolox/g extract). 2.10. Toxicological assessment 2.6. Microbial strains 2.10.1. Experimental animals Eleven strains of Pseudomonas aeruginosa ,1 24 strains of Thirty albino rats (170e200 g/rat) were used as an experimental Staphylococcus aureus ,2 11 strains of Aspergillus niger ,1 and 12 animal model for the in vivo oral toxicity assessment of the meth- strains of Candida albicans30 used in this study were isolated pre- anol extract of L. shawii leaves. The rats were kept for seven days viously from wound burn infections. As we previously described in prior to oral administration at the Animal House of Faculty of Sci- details these strains were identified as MDR or PDR strains.1,2,30 ence, Tanta University, Egypt to allow for their acclimatization to the laboratory environmental conditions as we previously 2.7. Antimicrobial activity, Minimum Inhibitory Concentration described in details.1,4 The animal experimental protocols were (MIC) and Minimum Fungicidal Concentration (MFC) performed in accordance with the requirements of the Institutional Animal Ethics Committee (IAEC) guidelines and approved by IAEC, Preliminary screening of antimicrobial activities of the four Faculty of Science, Tanta University, Egypt (code; Rec-Sci-Tu-020). L. shawii extracts was performed against 58 drug-resistant strains of bacteria and fungi (data not shown) using the disc diffusion 2.10.2. Acute oral toxicity 1 method as we previously described in details. According to our The toxicity test was designed to obtain the lethal dose 50 (LD50) 16 S.S. Ali et al. / Journal of Traditional and Complementary Medicine 10 (2020) 13e25 of L. shawii leaf extract according to the Organization for Economic necessary to reveal the biological activity and the chemical Cooperation and Development (OECD) Guideline.33 To determine composition as well as the safety assessment of the extracts from the LD50, rats divided into five groups, three rats in each. The the Egyptian L. shawii leaves. As depicted in Table 2, the aqueous, control group was administered with 0.5 mL saline solution, while methanol, ethanol and ethyl acetate extracts of L. shawii leaves other four groups were administered with methanol extract con- were evaluated for their phytochemical composition including al- centrations (1000e4000 mg/kg body weight) orally by gavage. Both kaloids, phenolics, flavonoids, tannins, saponins, glycosides, ter- control and treated groups were administered once daily for two penoids, steroids and coumarins. The results revealed that weeks. Visual observations including external appearance, body phenolics and flavonoids were found to be the most abundant weights, and daily activities were monitored throughout the compounds in all extracts tested. On the other hand, saponins and experimental period. coumarins were absent from all extracts. The high incidence of phytochemical compounds detected resulted in the superiority of 2.10.3. Haemolytic activity the methanol extract than that of the ethanol, ethyl acetate and The haemolytic activity assay of L. shawii extract was deter- aqueous extracts, respectively. Most of the conventional antibiotics mined as previously described.10 Briefly, the red blood cells were often fail to curb the proliferation of MDR/PDR clinical pathogens of collected from the healthy albino rats and the blood cell suspension bacteria and fungi in wound burn infections making the treatment was prepared by diluting the cell pellet to 0.5% with phosphate- of burns a difficult task. Plant extracts contain a diversity of buffered saline. Free haemoglobin of the supernatant was phytochemical compounds that used for the treatment of wound measured at 405 nm using a UVevisible spectrophotometer (Shi- burn infections since times immemorial. They also have a wide madzu-UV2600, Japan). Three replicates were done at each extract range of medicinal properties such as antibacterial, antifungal, concentration. Haemolytic activity was depicted by an increase in antioxidant, anti-inflammatory, antiallergic, antiproliferative, the optical density of the solution due to the release of hemoglobin antidiabetic, antiviral and analgesic that may prevent the devel- through time. opment of drug-resistant bacteria and fungi as reported for many traditional plant extracts.34,35 However, the information of L. shawii 2.11. Statistical analysis is limited. To the best of the authors' knowledge there have been no reports on the biological activities and the chemical composition of Results are presented as the mean ± standard deviation of three L. shawii leaves. Therefore, this study was aimed to assess the replicates. The statistical analyses were carried out using SPSS-20. antimicrobial and antioxidant activities of L. shawii leaves with The obtained data were analyzed statistically to determine the their toxicological evaluation. degree of significance using one-way analysis of variance and Student's t-test at a probability level 0.05. The Inhibitory Con- 3.2. Total bioactive compounds centration 50 (IC50) values were calculated by linear regression analysis. Linear regression was analyzed by Origin 6.0 professional As shown in Table 2, phenolics and flavonoids were found to be software. the most abundant phytochemical compounds in the L. shawii ex- tracts. Therefore, total quantities of these compounds were deter- 3. Results and discussion mined and results are given in Table 3. Phenolic compounds are associated with antioxidant activity. Hence, it is important to 3.1. Phytochemical screening of the L. shawii extracts quantify TPC and to assess its contribution to antioxidant activity. Clearly, the antioxidant activity of phenolic compounds is largely It is not enough to make a true assessment of the bioactivity associated with their redox potential which give them the superi- potential of a plant without considering its chemistry. The naturally ority to act as potential metal chelators, hydrogen donors, reducing occurring bioactive compounds are considered to be crucial for the agents as well as single oxygen quenchers.36 Our results of TPC biological activities of any plant species. Taken together, it is showed their wide variation in the four extracts tested, ranging
Table 2 Screening of phytochemical constituents of L. shawii leaf extracts.
Phytochemical Solvent extract Test or Observed Uses Mechanism of action References constituents reagent color Aqueous Methanol Ethanol Ethyl used acetate
Alkaloids ND þ þ ND Wagner Pale Antibacterial, anti-inflammatory, Intercalate into DNA and uncouple 34 yellow analgesic respiration 35 Flavonoids þ þ þ þ AlCl3 Yellow Antimicrobial, diuretic Metal chelating, cell wall complexation, adhesion binding 35 Phenolics þþ þ þ AlCl3 Yellow Antimicrobial, wound dressing Membrane infraction Tannins ND þ þ þ Braemers Dark blue Antimicrobial, wound dressing, Enzyme inactivation, cell wall complexation, 34 anti-inflammatory, preventing adhesion binding, membrane disruption, ulcer development substrate deprivation Saponins ND ND ND ND ND ee
Glycosides ND þ ND þ H2SO4 Dark Lower blood pressure NA brown Terpenoids ND þþþSalkowaski Red Antimicrobial Membrane disruption, prohibition of ATase 35 actuality Steroids ND þþND Salkowaski Red Antihypertensive effect on blood NA pressure, serum analysis of hypertensive patient Coumarins ND ND ND ND ND ee
ND, Not Detected; NA, Not Available; þ, present; -, absent; AlCl3, aluminum trichloride; H2SO4, sulfuric acid. S.S. Ali et al. / Journal of Traditional and Complementary Medicine 10 (2020) 13e25 17
Table 3 Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) of the solvent extract from L. shawii leaves.
Assay Solvent extract P-value
Aqueous Methanol Ethanol Ethyl acetate
TFC (mg QE/g extract)I 95.93 ± 0.64a 90.57 ± 0.31b 41.47 ± 0.51c 52.43 ± 0.98d < 0.001* TPC (mg GAE/g extract)II 55.50 ± 1.18a 230.50 ± 0.50b 99.93 ± 0.83c 190.60 ± 0.40d < 0.001*
Different superscript letters in the same row indicate significant difference (P 0.05); I QE, Quercetin Equivalent; II GAE, Gallic Acid Equivalent. from 55.50 ± 1.18 to 230.50 ± 0.50 mg GAE/g extract (Table 3). Ac- L. shawii leaf extracts as a remarkable source of antioxidant com- cording to these results, it is clear that the TPC depends on the type ponents. Accordingly, the antioxidant activities of these leaf ex- of extracted solvent used. The methanol extract had a significantly tracts are investigated in this study; especially there is no reports higher concentration of phenolics than ethyl acetate, ethanol and on the antioxidant activity of L. shawii leaves. aqueous extracts, respectively. This variation may be due to the phenolics polarity. Additionally, our findings may explain the 3.3. Antioxidant activity widespread uses of the folkloric plants. On the other hand, the TFC of the L. shawii extracts varied from 41.47 ± 0.51 to 95.93 ± 0.64 mg þ The DPPH and ABTS assays have been widely used to deter- QE/g extract (Table 3). The assay of TFC was resulted in the supe- mine the free radical-scavenging activity of various plant extracts. riority of the aqueous extract compared to the other extracts þ Both the DPPH (Fig. 1A) and ABTS (Fig. 1B) inhibition percentage including methanol, ethyl acetate and ethanol, respectively. Fla- values were dose-dependent, whereby they increased in the range vonoids, as a major class of phenolic compounds, are exhibited a of the tested concentrations, for the L. shawii extracts and the strong antioxidant activity. Clearly, our data of TPC and TFC suggest þ positive controls (GSH and Trolox for DPPH and ABTS ,