Antioxidant and Antimicrobial Potentials of Alkaloid- Rich Fraction of Zanthoxylum Zanthoxyloides’ Leaf Volume 52- Issue 5

Olusola AO¹, Olusola AO¹*, Elekan AO¹, Ogidan TO¹, Ekun OE¹ and Onoagbe IO²

• ¹Department of Biochemistry, Adekunle Ajasin University, Nigeria
• ²Department of Biochemistry, Faculty of Life Science, University of Benin, Nigeria

Received: September 08, 2023;   Published: September 20, 2023/p>

*Corresponding author: Olusola AO, Department of Biochemistry, Adekunle Ajasin University, Akungba Akoko, Ondo State, Nigeria

DOI: 10.26717/BJSTR.2023.52.008325

Abstract PDF

The need to scientifically prove the claims of the trado-medical uses of plants in combating diseases has led to research into the medicinal potencies of some plants. Zanthoxylum zanthoxyloides stem has been traditionally used to treat dental and malarial infections. This research was carried out to investigate the medicinal potentials in the leaf of this plant to seek its effectiveness against some diseases. In vitro antioxidant assays were carried out using 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), ferric reducing antioxidant power (FRAP), superoxide radical scavenging activity (SRSA) and hydrogen peroxide (H₂O₂) scavenging activity. Ascorbic acid was used as standard antioxidant. In vitro antimicrobial sensitivity testing was also done using oral microbes obtained from human sputum. Ciprofloxacin was used as positive control, while methanol was the negative control. The minimum inhibitory concentrations for the microbes were determined for K. pneumonia at 100 mg/ml, S. aureus at 50 mg/ml and M. catarrahlis at 50 mg/ml. The EC₅₀ values, the concentration that gives half maximal response of the extract to scavenge the reactive oxygen species, were obtained for DPPH as 0.6043±0.0014 mg\ml, H₂O₂ as 0.7277±0.0339 mg\ml and SRSA as 0.0770±0.0075 mg\ml.  Antimicrobial testing also showed the extract was sensitive to Staphylococcus aureus, Moraxella catarrhalis and Klebsiella pneumoniae at concentrations of 500 mg/ml, 250 mg/ml, 125 mg/ml and 100 mg/ml. Evidently, the alkaloid-rich extract of Z. zanthoxyloides possesses potent antioxidant and antimicrobial principles, which lay credence to the traditional use of the plant in the treatment of dental and malarial infections. Consequently, the use of the plant in traditional medicine should therefore be encouraged.

Keywords: Z. Zanthoxyloides; Alkaloid-Rich Fraction; Antioxidant; Antimicrobial

Abbreviations: FRAP: Ferric Reducing Antioxidant Power; SRSA: Superoxide Radical Scavenging Activity; As: Absorbance value of Test Samples; Ac: Absorbance of Hydrogen Peroxide; FUTA: Federal University of Technology Akure; S: Sensitive; I: Intermediate; R: Resistant

Medicinal herbs have been used for healing as an alternative to medicine by all cultures for several thousands of years. About 80% of the world’s population does not have access to conventional drugs and therefore rely on medicinal herbs (Abugassa, et al. [1]). In Nigeria, Zanthoxylum zanthoxyloides is used as a chewing stick. Water extracts from the plant showed activities against bacteria significant to periodontal disease (Taiwo, et al. [2]). The anthelmintic activity of the methanolic extract of the root-bark of Z. zanthoxyloides was also reported (Ogwal-Okeng, 1999), and it is a very popular anthelmintic amongst the various tribes in Uganda. It has also been found that the alcoholic extracts of the root-bark possesses considerable antibacterial activity (El-Said et al. [3]).  Several studies on the various effects of its extracts have been reported. For example, Kassim, et al. [4] reported the anti-malarial activity attributed to benzophenanthridine alkaloid, fagaronine from Z. zanthoxyloides root extracts. Anti-malarial activity was also reported in a study using extracts from trunk barks of Z. zanthoxyloides (Gansane et al. [5]). On the other hand, Patel et al. [6] named the compound nitidine as the agent in Z. zanthoxyloides’ anticancer capabilities while an anti-inflammatory property due to ortho-hydroxymethyl benzoic acid made Z. zanthoxyloides useful in the management of pain in sickle cell crisis (Oyedapo, et al. [7,8]). More recently, the potential of Z. zanthoxyloides leaf, bark and root extracts as a biopesticide for stored food protection has been reported (Udo [9]).

Zanthoxylum zanthoxyloides leaves were obtained from a farm in Ikare Akoko area of Ondo state, Nigeria. The leaves were identified at the Department of Plant science and Biotechnology, Adekunle Ajasin University, Akungba-Akoko, Ondo state, Nigeria. The leaves were washed and air-dried for 8weeks, then it was blended into fine powder. The powdered leaves were soaked with methanol at ratio 5:1(500 ml of methanol to 100 g of the sample) for 72 hours and a clean muslin cloth, was used to filter the soaked sample, the filtrate was then evaporated using the rotary evaporator at 40^oC. The resulting solution was then air dried and kept in the refrigerator at -4^oC until use.

Extraction of the Alkaloids-Rich Fraction

To 800 g of the plant sample, 4 litres of methanol was added and macerated for 72 hrs and then sieved with a clean muslin cloth. After filtration, the solvent was removed under reduced pressure using a rotary evaporator at 40⁰C, to minimize any thermal degradation of the alkaloids. Alkaloid was extracted using the procedure described by Hadi and Bremner [10]. The crude alkaloid mixture was then separated from neutral and acidic materials and water solubles, by initial extraction with aqueous acetic acid, followed by dichloromethane extraction and then alkanation of the aqueous solution and further dichloromethane extraction.

Determination of DPPH Radical Scavenging Activity

The DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging activity of Alkaloid-rich leaf extract of Zanthoxylum zanthoxyloides was measured by using assay method described by Shimada et al. [11] with slight modifications. 1 mL each of plant extract at different concentrations (0.2 – 0.8 mg/ml) was added to 1 mL 0.1 mM DPPH dissolved in 95 % ethanol. The mixture was shaken vigorously and incubated in the dark and at room temperature for 30 min. The absorbance was read at 517 nm. Ethanol (95 %) was used as a blank. The control solution consisted of 0.1 mL of 95 % ethanol and 2.9 mL of DPPH solution. Analyses were carried out in triplicates. Percentage inhibition of DDPH radical was calculated.

IC₅₀ values were estimated from percentage inhibition plot, using a non-linear regression plot.

Determination of Hydrogen Peroxide Scavenging Activity

The ability of the Alkaloid-rich leaf extract of Zanthoxylum zanthoxyloides to scavenge hydrogen peroxide was determined according to the method of Ruch et al. [12] as described by Keser et al. [13] with slight modifications. A solution of hydrogen peroxide (4 mM) was prepared in 0.2 M phosphate buffer (pH 7.4). Varying concentrations of the extract (0.2 – 0.8 mg/mL) in distilled water were added to 0.6 ml of 4mM hydrogen peroxide solution. Absorbance value of test samples (As) were read at 230 nm after 10 minutes against a blank solution containing the phosphate buffer without hydrogen peroxide. Absorbance of hydrogen peroxide (Ac) was taken as the control. Ascorbic acid was used as a standard antioxidant. The percentage of scavenging effect was calculated by comparing the absorbance values of the control and test samples using:

IC₅₀ values were estimated from the % inhibition versus concentration plot, using a non-linear regression plot.

Determination of Ferric Reducing Antioxidant Property (FRAP)

The reducing power of alkaloid-rich leaf extract of Zanthoxylum zanthoxyloides was measured according to the method of Oyaizu [14] with slight modification. An aliquot of 1 ml of different concentrations (0.2 – 0.8 mg/ml) of alkaloid-rich leaf extract (0.2 M PBS, pH 6.6) was mixed with 1 ml of 1% potassium ferric cyanide solution. The mixture was incubated at 50°C for 30 minutes followed by the addition of 1 ml 10% (w/v) TCA. 1 ml of the incubation mixture was added with 1 ml of distilled water and 0.2 ml of 0.1% (w/v) ferric chloride in test tubes. After a 10 min reaction time, the absorbance of resulting solution was read at 700 nm. Higher absorbance suggested stronger reducing power. Ascorbic acid was used as the reference antioxidant. An aqueous solution of known Fe (II) concentrations (FeSO₄·7H₂O; 2.0, 1.0, 0.5, 0.25, 0.125, 0.063 mM) was used for calibration. Results were expressed as mM Fe²⁺/mg extract. All the tests were performed in triplicate. The EC₅₀ of extracts were calculated from the graph of A700 versus extracts concentration.

Determination of Superoxide Radical Scavenging Activity

The method described by Xie et al., (2008) was used to determine SRSA. Samples (1 mg/mL final concentration) were each dissolved in 50 mM Tris–HCl buffer, pH 8.3 containing 1 mM EDTA and 80 μL was transferred into a clear bottom microplate well; 80 μL of buffer was added to the blank well. This was followed by addition of 40 μL 1.5 mM pyrogallol (dissolved in 10 mM HCl) into each well in the dark and the change in the rate of reaction was measured immediately at room temperature over a period of 4 min using a spectrophotometer at a wavelength of 420 nm. The superoxide scavenging activity was calculated using the following equation:

where b and s are blank and sample, respectively.

Susceptibility of bacteria and fungi isolate to plant extract was determined following the BSAC Diffusion Method for Antimicrobial Susceptibility Testing Version 8 (Andrews [15]). This test was carried out to determine the antimicrobial ability of the   plant extract to inhibit the growth of the bacteria and fungi isolate that was collected from Microbiology Department of Federal University of Technology Akure (FUTA). The plate diffusion technique of Willey et al. [16] was used for the antibiotic sensitivity test. Overnight cultures of the organisms were swabbed on sterile Muller Hilton solidified Agar plates using sterile swab sticks. 8mm sized cork borer was used to bore hole on the agar surface at equidistance the well was filled with the diluted plant extract, a known antibiotic was used as positive control while distilled water was used as negative control. All the plates were incubated at 37⁰C to 24 hours. The zones of inhibition generated by the antibiotics were measured to the nearest millimetres (mm) and interpreted as sensitive (S), Intermediate (I) and resistant (R). The zones of inhibition were measured and interpreted according to (NCCLS, 2000).

Statistical Analysis

Results were expressed as mean of replicates ± standard error of mean. The data were statistically analyzed using t-test and Duncan’s multiple range tests. Differences were considered statistically significant at p<0.05 using SPSS and GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA, USA).

Antioxidant Results for Alkaloids

DPPH Radical Scavenging Activity (%)

DPPH EC₅₀

Bars are expressed as means ± standard error of mean of triplicate determinations (n=3). Values with the same superscripts do not differ significantly while values with different superscripts are significantly different (p<0.05) from one another.

 Hydrogen Peroxide Scavenging Activity (%)

H₂O₂ EC₅₀ (mg/ml): Bars are expressed as means ± standard error of mean of triplicate determinations (n=3). Values with the same superscripts do not differ significantly while values with different superscripts are significantly different (p<0.05) from one another.

Ferric Reducing Antioxidant Power (FRAP) (mmol Fe²⁺): Figure 1 shows the ferric reducing antioxidant properties of ascorbic acid (control) and the alkaloid rich extract of zanthoxyloides. It significantly (p<0.05) reduced antioxidant activity which is concentration dependent when compared to ascorbic acid.

Figure 1

Note: Bars are expressed as means ± standard error of mean of triplicate determinations (n=3). Values with the same superscripts do not differ significantly while values with different superscripts are significantly different (p<0.05) from one another.

Bars are expressed as means ± standard error of mean of triplicate determinations (n=3). Values with the same superscripts do not differ significantly while values with different superscripts are significantly different (p<0.05) from one another.

Superoxide Radical Scavenging Activity (%)

Superoxide Radical EC₅₀ (mg/ml)

Antimicrobial Assay Results

(Tables 1-3).

Table 1: Result of antibacterial test using alkaloids-rich extracts of Z. zanthoxyloides against some bacteria.

Note: Sensitive (S) ≥ 21, Intermediate (I) 20≤ 15 and resistant (R) ≤ 14

Table 2: Result of antifungal test using alkaloid-rich extracts of Z. zanthoxyloides against some fungi.

Note: Sensitive (S) ≥ 21, Intermediate (I) 20≤ 15 and resiq stant (R) ≤ 14

Table 3: Result of minimum inhibitory concentration test using alkaloid-rich extracts of Z. zanthoxyloides against some bacteria.

Note: Sensitive (S) ≥ 21, Intermediate (I) 20≤ 15 and resistant (R) ≤ 14

DPPH Radical Scavenging Activity of Alkaloid-Rich Fraction of Zanthoxyloides

DPPH (2,2-diphenyl-2-picrylhydrazyl) analysis is one of the best known, accurate, and frequently employed methods for evaluating antioxidant activity.  It is a stable free radical which determines the ability of pure substances or crude extracts for trapping the unpaired electron species by donating hydrogen atoms or electrons, and produced in consequence of the radical disappearance and the formation of less reactive species derived from the antioxidant (Parekh et al. [17]). In this study, the effect of the alkaloid-rich extract of Z. zanthoxyloides on DPPH scavenging activity increased, with the increase in concentration of the extract from 0.2-0.8 mg\ml and it is thought to be due to their hydrogen donating ability. The DPPH radical scavenging activities of the alkaloid-rich extract as well as their EC₅₀ values in comparison to ascorbate (standard antioxidant) are as presented in Figures 2 & 3 respectively. These results appear to be similar to that obtained by Suman et al. [18] for an aporphine alkaloid crebanine derived from Alphonsea sclerocarpa. The EC₅₀ values show that the extract scavenged the radical to a 50% inhibition at a concentration of 0.6043±0.0286 mg/ml.

Figure 2 DPPH Scavenging Activities of alkaloid-rich extract of Z.zanthoxyloides.

Note: Each point represents an average of triplicate determinations (n=3)

Figure 3 Hydrogen peroxide scavenging activity of alkaloid-rich extract of Z.zanthoxyloides.

Note: Each point represents an average of triplicate determinations (n=3)

Hydrogen Peroxide Scavenging Activity of Alkaloid-Rich Fraction of Zanthoxyloides

Hydrogen peroxide (H₂O₂) is a weak oxidizing agent and crosses cell membranes rapidly to enter the cell. There, H₂O₂ can react with Fe⁺² ions to generate the hydroxyl radical and this may be the origin of many of its toxic effects (Halliwell, et al. [19]). H₂O₂ is relatively stable in the absence of reducing compounds. Scavenging of H₂O₂ by the extract may be attributed to their electron donating abilities (Wettasinghe, et al. [20]). The H₂O₂ scavenging activity of alkaloid-rich extract of Z. zanthoxyloides and their EC₅₀ values are presented in Figures 4 & 5 respectively. The result revealed a higher antioxidant activity of the extract at concentrations 0.2 and 0.4 mg\ml greater than the standard and at concentrations 0.6 and 0.8 mg\ml, the scavenging activity was significantly reduced compared to that of the standard. The extract scavenged the radical to a 50% inhibition at a concentration of 0.7277 ±0.0339. The ability of bioactive compounds to reduce ferric ions has a strong correlation with their antioxidative properties. FRAP method is based on the reduction of Fe³⁺ to Fe²⁺ by antioxidants in acidic medium (Benzie, et al. [21]). The FRAP activity of the alkaloid-rich extract is presented in Figure 1. The radical scavenging activity of the extract is concentration dependent, the scavenging activity increased with increase in the extract concentration. This result was found to be similar to that obtained by Suman et al. [18] for a aporphine alkaloid crebanine derived from Alphonsea sclerocarpa.

Figure 4 DPPH Radical EC₅₀ values alkaloid-rich extract of Z. zanthoxyloides.

Note: Bars are expressed as means ± standard error of mean of triplicate determinations (n=3). Values with the same superscripts do not differ significantly while values with different superscripts are significantly different (p<0.05) from one another.

Figure 5 Hydrogen peroxide radical EC50 values of alkaloid-rich extract of Z.zanthoxyloides.

Note: Bars are expressed as means ± standard error of mean of triplicate determinations (n=3). Values with the same superscripts do not differ significantly while values with different superscripts are significantly different (p<0.05) from one another.

Superoxide Radical Scavenging Activity of Alkaloid-Rich Fraction of Zanthoxyloides

Superoxide dismutase catalyses the dismutation of the highly reactive superoxide anion to oxygen and hydrogen peroxide. These species are producewd by a number of enzyme systems in auto-oxidation reactions and by non-enzymatic electron transfers that reduces molecular oxygen. It can also decrease certain iron complexes such as cytochromes (Sena et al. [22] Gulein et al. 2011). (Korycka-Dahl, et al. [23]). The superoxide activity of alkaloid-rich extract of Z. zanyhoxyloides and their EC₅₀ values were presented in Figures 6 and 7 respectively.  This study reveals that the alkaloid-rich extract showed a potent superoxide scavenging activity but lower to that of ascorbic acid. The extract scavenged the radical to a 50% inhibition at a concentration of 0.0770±0.0075 mg\ml. The presence of secondary metabolites like flavonoids, carotenoids and triterpenes in higher plants have excellent antioxidant activity by scavenging reactivity oxygen species which prevent possible damage to cellular components such as DNA, proteins and lipids (Ksouri et al. [24]) [25-35].

Figure 6 Superoxide scavenging activity of alkaloid-rich extract of Z.zanthoxyloides.

Note: Each point represents an average of triplicate determinations (n=3).

Figure 7 Superoxide radical EC₅₀ values of alkaloid-rich extract of Z.zanthoxyloides.

Note: Bars are expressed as means ± standard error of mean of triplicate determinations (n=3). Values with the same superscripts do not differ significantly while values with different superscripts are significantly different (p<0.05) from one another.

Antimicrobial Assay

Considering the results obtained above, it was confirmed that at 20 mg -40 mg the extract did not show sensitivity to any of the five bacteria on which it was tested. At 60 mg, the alkaloid-rich extract was not sensitive to the bacteria on which it was tested. At 80 mg, the bacteria Klebsiella pnuemoniae and Citrobacter amalonaticus were not sensitive to the alkaloid-rich extract while the extract was resistant to Staphylococcus aureus, Bacillus subtillis and Escherichia coli. At 100 mg, the K. pnuemoniae and C. amalonaticus were resistant to the alkaloid-rich extract while the S. aureus, B. subtillis and E. coli were intermediately sensitive. The minimum inhibitory concentration of the alkaloid-rich extract for S. aureus, B. subtillis and E. coli is 100 mg. The extract showed intermediate inhibitory activity against Aspergillus favus, Rhyzopus stolonifer and Fusarium solani while it was resistant against the Candida tropicalis and Candida albican.

The alkaloid-rich extract showed potent antioxidant properties comparable to those of the synthetic antioxidant. Thus, the fraction can be utilized as a natural source of antioxidant.

1. Abugassa IO, Bashir AT, Doubali K, Elwir RH, Abu-Enavel M, et al. (2008) Characterisation of Trace Elements in Medicinal Herbs by Instrumental Neuron Activation Analysis. Journal of Radioanalogy and Nuclear Chemistry 278(3): 559-563.
2. Taiwo O, Xu HX, Lee SF (1999) Antibacterial activities of extracts from Nigerian chewing sticks. Phytother Res 13(8): 675-679.
3. El-Said F, SO Fadulu JO Kuye, Sofowora EA (1971) Native cures in Nigeria: II The antimicrobial properties of the buffered extracts of chewing sticks. Lloydia 34: 172-174.
4. Kassim OO, Loyevsky M, Elliott B, Geall A, Amonoo H, et al. (2005) Effects of root extracts of Zanthoxyllum zanthoxyloides on the in vitro growth and stage distribution of Plasmodium falciparum. Antimicrobial Agents and Chemotherapy 49(1): 264-268.
5. Gansané A, Sanon S, Ouattara LP, Traoré A, Hutter S, et al. (2010) Antiplasmodial activity and toxicity of crude extracts from alternative parts of plants widely used for the treatment of malaria in Burkina Faso: contribution to their preservation. Parasitology Research 106(2): 335-340.
6. Patel B, Das S, Prakash R, Yasir M (2010) Natural bioactive compound with anticancer potential. Int J Adv Pharm Sci 1(1): 32-41.
7. Oyedapo OO, Famurewa AJ (1995) Antiprotease and membrane stabilizing activities of extracts of Fagara. Zanthoxyloids, Olax subscorpioides and Tetrapleura tetraptera. Pharmacol Biol 33(1): 65-69.
8. Folasade SI, Olukemi OA, Jones MO (2006) Management of sickle cell anemia in Nigeria with medicinal plants: Cationic evaluation of extracts and possible effects on the efficacy. J Biol Sci 6(1): 100-102.
9. Udo IO (2011) Potentials of Zanthoxylum xanthoxyloides (LAM.) for the control of stored product insect pests. J Stored Prod Postharvest Res 2(3): 40-44.
10. Hadi S, Bremner JB (2001) Initial studies on alkaloids from Lombok medicinal plants. Molecules 6(2): 117-129.
11. Shimada K, Fujikawa K, Yahara K, Nakamura T (1992) Antioxidative properties of Xanthan on the antioxidation of soybean oil in cyclodextrin emulsion. J Agric Food Chem 40: 945-948.
12. Ruch RJ, Cheng SJ, Klaunig JE (1989) Prevention of cytotoxicity and inhibition of intracellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis 10(6): 1003-1008.
13. Keser S, Celik S, Turkoglu S, Yilmaz O, Turkoglu I (2012) Hydrogen Peroxide Radical Scavenging and Total Antioxidant Activity of Hawthorn. Chemistry Journal 2(1): 9-12.
14. Oyaizu M (1986) Studies on products of browning reactions: antioxidative activities of 581 products of browning reaction prepared from glucosamine. Jpn J Nutr 44(582): 307-315.
15. Andrews JM (2009) BSAC standardized disc susceptibility testing method (version 8). Journal Antimicrob Chemother 64(3) :454-489.
16. Willey JM, Sherwood LM, Woolverton CJ (2008) Prescott, Harley and Klein’s Microbiology (7^th (dn.)., NY, McGraw Hill.
17. Parekh J, Darsharna J, Chanda S (2007) Efficacy of aqueous and methanol extracts of some medicinal plants for potential antimicrobial activity. Turkish Journal of Biology 29: 203-210.
18. Suman Joshi DSD, Chander Amgoth Surya Narayana S, Padmavathi CH, Madhavi J, Krisha Satya A (2018) Antioxidant and anticancer activities of an Aporphine alkaloid isolated from Alphonsea sclerocarpa. 7(1): 51-55.
19. Halliwell B, Gutteridge JMC (1989) Free Radicals in Biology and Medicine. Second Edition. Clarendon Press Oxford.
20. Wettasinghe M, Shahidi F (2000) Scavenging of reactive-oxygen species and DPPH free radicals by extracts of borage and evening primrose meals. Food Chemistry 70(1): 17-26.
21. Benzie IFF, Strain JJ (1996) The Ferric Reducing Ability of Plasma (FRAP) as a measure of antioxidant power. The FRAP assay. Anal. Biochem 239(1): 70-76.
22. Sena IM, Zacolotto SM, Schenkel EP, De Lima TC (2009) Neuropharmacological activity of the pericarp of Passiflora edulis flavicarpa degener: putative involvement of C-glycosyl flavonoids. Experimental Biology and Medicine 234(8): 967-975.
23. Korycka-Dahl, M, Richardson T (1978) Photogeneration of superoxide anion in serum of bovine milk and in model system containing riboflavin and amino acids. Journal of dairy science 61(4): 400-407.
24. Ksouri WM, Medini F, Mkadmini K, Legault J, Magne C, et al. (2013) LC-ESI-TOF-MS identification of bioactive secondary metabolites involved in the antioxidant, anti-inflammatory and anticancer activities of the edible halophyte Zygophyllum album Desf. Food Chem 139(1-4): 1073-1080.
25. Abalaka MZ, Mann A, Adeyemo SO (2011) Studies on in-vitro antioxidant and free scavenging potential and phytochemical screening of leaves of Ziziphus mauritiana L. and Ziziphus spinachristi L. compared with Ascorbic acid. Journal of Medical Genetics and Genomics 3(2): 28-34.
26. Gulein I, Alici HA, Cesur M (2005) Determination of in vitro antioxidant and radical scavenging activities of propofol. Chemical and pharmaceutical Bulletin 53(3):281-285.
27. (2000) NCCLS. Performance standards for antimicrobial disk susceptibility tests. Approved standards in (7^th)., NCCLS document M2-A7. NCCLS, Wayne Pa.
28. Ogwal-okeng JW, Obua C, Anokbonggo WW (2003) Acute toxicity effects of themethanolic extract of Zanthoxyllum zanthoxyloides (Lam.) root-bark. African health Sciences 3(3): 124-126.
29. Olusola AO, Ogunsina OI, Olusola AO (2020) Antimalarial potential of flavonoid-rich extract of Lannea acida and chloroquine in mice infected with Plasmodium berghei. Int Journal of Scientific and Engineering Research 11(3): 201-206.
30. Sahoo S, Goutam G, Das D, Nayak S (2013) Phytochemical investigation and in vitro antioxidant activity of an indigenous medicinal plant activity of an indigenous medicinal plant Alphina nigra B.L. Burtt. Asian Pac J Trop Biomed 3(11): 871-876.
31. Sofowora EA, WA Isaac-Sodeye, LO Ogunkoya (1975) Isolation and characterization of an antisckling agent from Fagara zanthoxyloides Root. Lloydia 38(2): 169-174.
32. Sumazian Y, Syahida A, Hakiman M, Maziah M (2010) Antioxidant activities, flavonoids, ascorbic acid and phenolic contents of Malaysian vegetables. Journal of Medicinal Plants Research 4(10): 881-890.
33. Xie Q, Johnson BR, Wencku CS, Fayad MI, Wu CD (2012) Efficacy of berberine, an antimicrobial plant alkaloid, as an endodontic irrigant against a mixed culture biofilm in an in vitro tooth model. J Endod 38(8): 1114–1117.
34. Yildirim A, Mavi A, Oktay M, Kara AA, Algur OF, et al. (2000) Comparison of antioxidant and antimicrobial activities of tilia (Tilia argenta Desf. Ex DC), sage (Salvia triloba L.) and black tea (Camellia sinensis) extracts. J Agr Food Chem 48(10): 5030-5034.
35. Zhao H, Wang Z, Cheng C, Yao L, Wang L, Lu W, et al. (2011) In-vitro free radical scavenging activities of anthocyanins from three berries. Journal of Medicinal Plants Research 5(32): 7036-7042.