ANTIMICROBIAL AND ANTIOXIDANT ACTIVITY OF PSIDIUM GUAJAVA. (GUAVA) MEDICINAL PLANT LEAVES USED IN FOLK  MEDICINE FOR TREATMENT OF  WOUNDS  AND BURNS  IN HUFASH DISTRICT AL MAHWEET GOVERNORATE–YEMEN

Samir Ahmed Ali ALhaidari¹, Aziza M. Taj Al-Deen¹, Ali Gamal Al Kaf², 

  Fatima A. Al-Hadi¹, Qais Abdullah¹, Anas AL Mahbashi¹, Fouad A. Al-Dubai³     

¹Biology Department -Faculty of Science, Sana'a University, Yemen.              

²Medicinal chemistry Department, Faculty of pharmacy, Sana'a University,Yemen.                             

³Biology Department, Faculty of science, Dhamar University, Yemen.

DOI: https://doi.org/10.22270/ujpr.v4i2.250

ABSTRACT

In this study methanolic and aqueous extracts of one plant namely Psidium guajava, were screened for the presence of phytochemical constituents and tested for their antimicrobial and antioxidant activity. The qualitative phytochemical analysis revealed the results showed presence of alkaloids, terpenoids, glycosides, resins, saponins, tannins, flavonoids, phenols, and amino acid were present in the methanol extract, with absence of glycosides, and amino acids in the aqueous extracts in leaves plant. TLC tests conducted revealed Rf values in the leaves for alkaloids, Flavonoids, Tannins, Phenols and Saponins(0.96-0.97-0.99-0.97-0.99) respectively. The antimicrobial activity extracts against four bacterial isolates Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella sp. and a single fungal isolate Candida albicans with concentrations (0.5 mg/ml, and 1,0 mg/ml) of the extract were added to the disc and respective solvent was used as negative control. The antioxidative activity of leaf was evaluated by using 1,1-  diphenyl-2 picrylhydrazyl (DPPH), the results showed are 88.4%, highest from standard, ascorbic acid 87.5%.

Keywords: Antimicrobial, antioxidative, phytochemical, Psidium guajava.

INTRODUCTION

Psidium guajava (PG) belongs to the family Myrtaceae, which is considered to have originated in tropical South America. Guava crops are grown in tropical and subtropical areas of the world like Asia, Egypt, Hawaii, Florida, Palestine and others. The genus Psidium comprises approximately 150 species of small trees and shrubs in which only 20 species produce edible fruits and the rest are wild with inferior quality of fruits¹. Psidium guajava is a phytotherapic plant used in folk medicine that is believed to have active components that help to treat and manage various diseases. The many parts of the plant have been used in traditional medicine to manage conditions like malaria, gastroenteritis, vomiting, diarrhea, dysentery, wounds, ulcers, toothache, coughs, sore throat, inflamed gums, and a number of other condition². Psidium guajava leaves extract has a wide spectrum of biological activities such as anticough, antibacterial, haemostasis, anti diarrhoeal and narcotic properties³. The leaves of guava contain an essential oil rich in cineol, tannins, triterpenes, flavonoids, resin, eugenol, malic acid, fat, cellulose, mineral salts, and a number of other fixed substances⁴. In a study that attempted to investigate antioxidant activity of Psidium guajava leaves extract by DPPH 2, 2- diphenyl-1-picrylhydrazyl) free radical scavenging method using ascorbic acid as standard, it was found that the extract of P. Guajava leaves extract was found to possess strong antioxidant activity ,this activity of P. Guajava extract may be attributed to their free radical-scavenging ability. The extent of antioxidant activity of P. Guajava extract was found significant as compared to standard value for P. Guajava linn leaves extract was found to be 45.5 μg/ml. Thus P. Guajava linn leaves possess moderate antioxidant activity as compared as standard⁵.

MATERIALS AND METHODS

Samples extraction: The Samples of 100g of the grinded powder were put in sterilized flasks together with 400 ml of pure methanol for methanolic extraction treatments, while for aqueous extraction treatments, samples of 100g of grinded powder were put in sterilized flasks with 400 ml of distilled water each. All flasks were covered with transparent nylon and tin and then all were put on a rotary shaker machine for 24 hours. The filtration process for each sample was carried out using filter paper to obtain a pure solution. The evaporation process for each methanol solution and distilled water was conducted separately in the evaporator methanol and distilled water solution. Then the obtained extracts were kept in dark conditions in the refrigerator at 4°C until used in the experiment⁶.

Qualitative tests                   

Phytochemical screening of plant extracts

The methanolic and aqueous extracts subjected to phytochemical screening were   alkaloids, terpenoids, glycosides, resins, saponins, tannins, flavonoids, phenols, and amino acids.

Alkaloids: Dragendorff’s test

In a test tube, 2-3 drops of Dragendorff’s reagent was added to 0.1 ml of the extract ,orange precipitate indicated the presence of alkaloids.

Terpenoids: Salkowski test

In a test tube 5ml of extract was mixed in 2ml of chloroform and then 3ml of concentrated sulfuric acid was added to form a layer. A reddish brown coloration forms at interface.

Glycosides: Keller-Killani test

Concentrated sulfuric acid in a test tube and extract sample were mixed with glacial acetic acid containing 1 drop of Ferric chloride (1:1:1volume). A brown ring appears in the presence of glycosides.

Resins: Turbidity test

To 5ml extract 5ml distilled water was added, the occurrence of turbidity shows the presence of resins.                  

Saponins: Foam test

A 5ml extract was shaken with 2 ml of distilled water. If foams are produced and persists for ten minutes this indicates the presence of saponins.

Tannins: Fecl₃ test

A 4 ml extract was treated with 4 ml FeCl₃, the formation of green colour was taken as positive for tannin.

Flavonoids: Shinoda test

Extract was mixed with magnesium ribbon fragments, and concentrated hydrochloric acid was added drop wise. Orange, red, pink, or purple coloration indicates the presence of flavonoids.

Phenols: Fecl₃ test

Extract was mixed with 2 ml of 2% solution of FeCl₃. A blue-green or black coloration indicated the presence of phenols.

Amino acids: Biuret test

Extracts and 1 drop 2% Copper sulphate solution and 1 ml 95% ethanol excess of potassium hydroxide were mixed. Pink or yellow color in ethanol layer appears.

Thin Layer Chromatography.

One gram of Psidium guajava, powder was boiled with of with solvent system made from 15ml H₂SO₄ test for Alkaloids_, 10ml 70% ethanol test for Flavonoides and Saponins, 25 ml water test for Tannins and phenols in rounded flasks. The TLC plate was prepared as such : (Layer: silica gel layers 0.25 mm thickness, 10 cm length and 5cm wide). The filtrate obtained was evaporated to dryness in a water bath at 37° C .The residue was dissolved by 0.2ml methanol. The solution was used for spotting the TLC by capillary tube by only one centered spot. The TLC plate was put inside a saturated tank, and development was waited. When the mobile phase reaches two thirds of plate^’s length, the plate was lifted out from the tank and let to dry in air. The plate was examined by U.V. lamp at the wavelength 365nm.  The colors of florescence appeared and recorded. The plate was sprayed carefully reagent, and let to dry for 10 min. Then spray et with solution .Then plate was examined under U.V. lamp at the wavelength 365nm. The iodine was used as the visualizing agent to detect the spot. A meter rule was used to measure the distance moved by the solvent and distance moved by spot, from which the retention factor (R_f values) of the various spots was calculated⁶.  TLC was performed for alkaloids, flavonoids, tannins and phenols solvent system and confirmatory tests are shown in Table 2.                             

Calculation of RF of each spot was as follows

Antimicrobial Activity of Plants extracts.

Microbial Cultures: Fresh plates of the four bacterial isolates Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella sp. and a single fungal isolate Candida albicans were obtained from the National Center of Public Health Laboratories, Sana’a.

Media Use: The bacterial test were spread over the nutrient ager (56g/1000ML distilled water) was weight into separate flask and dispensed into distilled water make a total volume of 1 liter. Then the fungal test were spread over the sabouraud dextrose ager (65g/1000ML distilled Water) was weighted into separate flask and dispensed into distilled water to make a total volume of 1 liter. These powders were dissolved in distilled water and used for evaluation of their antibacterial and antifungal activities. The mixture was heated in an electric water bath (GFC, 1083, Germany) until the Agar melted to form a homogenous solution. The prepared medium was separately transferred to Durum medium bottle and sterilized by autoclaving at 121⁰ C for 30 minutes. The sterile medium was allowed to cool to about 45^oC before being poured aseptically in an inoculation chamber (Ceslab England) in15 ml portions, into sterile petri dishes to cool and gel into solids⁸.  

Antimicrobial activity assay: Two different concentrations (0.5 mg/ml, and 1.0 mg/ml) of the extract were added to the disc and respective solvent was used as negative control.

Zone of inhibition:  The bacteria plates were incubated at37^oC for 24hrs while the fungal   plates were incubated at for 72 hours, and observed for the zone of inhibition of growth. The zones were measured with a transparent ruler and the result recorded.                                                                       

Determination of antioxidant activity

The scavenging ability of the natural antioxidants of the leaves towards the stable free radical DPPH was measured by the specific method⁹. The leaf extracts (20μl) were added to 0.5ml of methanolic solution of DPPH (0.3mM in methanol) and 0.48ml of methanol. The mixture was allowed to react at room temperature for 30 min. Methanol served as the blank and DPPH in methanol, without the leaf extracts, Served as the positive control. After 30 min of incubation, the discolouration of the purple colour was measured at 517 nm in a spectrophotometer). The radical scavenging activity was calculated as follows:

Radical Scavenging Activity

Statistical Analysis

Analysis of variance was made for all data using (SPSS) version (25) computer program.

RESULTS AND DISCUSSION

In this study methanolic and aqueous extracts of one plants namely Psidium guajava, were screened for the presence of phytochemical constituents and tested for their microbial and antioxidant activity.

Yield from different solvents                                                  

Yield of methanolic extract of Psidium guajava, extracted with 100% methanol produced 32.40(g). While yield of distilled water extract of Psidium guajava produced 27.62(g). Mean values of the yield are presented as mean ± SEM. Values are statistically   significant when p≤ 0.05. A similar investigation done in a study¹⁰ revealed that aqueous extracts (16.35%) of Psidium guajava gave high yields than of methanolic extracts (14.22%), which is contrary to our findings. Similarly, a previous study also reported a 16.35% yield in aqueous extracts from Psidium guajava¹¹. Yet the percentages of yields in both studies were less than of the present study.

Phytochemical composition of the methanolic and aqueous leaves   extracts.     

The summarized phytochemical screening of chemical constituents of Psidium guajava extract is shown in Table 3. The results revealed the presence of active compounds in the two different extracts. As the table shows, the methanol and aqueous extracts indicate the presence alkaloids, terpenoids, glycosides, resins, saponins, tannins, flavonoids, phenols, and amino acid were present  in the methanol extract, with absence of glycosides, and amino acids in the aqueous extracts in all three plants .In a previous study, methanolic extracts of Psidium guajava revealed the presence of alkaloids, tanins, flavonoids and glucosides¹². Similary other study has  showed the presence of tannins, saponins, flavonoids, alkaloids and phenols as our study¹³.         

Thin Layer Chromatography (TLC)

Five secondary metabolites (alkaloids, flavonoids , tannins, phenols and  saponins) were used  for  (TLC) thin layer chromatographic analysis. TLC tests conducted revealed R_f values in the leaves of Psidium guajava for alkaloids, Flavonoids, Tannins, Phenols and Saponins(0.96-0.97-0.99-0.97-0.99) respectively. In a study done through TLC profiling proved that different Rf values represent different chemical constituents present within methanol leaf extract of Psidium guajava¹⁴. There were six visible spots. The Rf values (spot 1, RF=0.98), (spot 2, RF=0.78), (spot 3, RF=0.62), (spot 4, RF=0.54) (spot 5, RF=0.32) and (spot 6 RF=0.19). Similar R_f values were in agreement with this investigation.                                                                                                                                                                

Antibacterial and antifungal activity of plants extracts

Antimicrobial activity of standard antibiotics discs against tested bacterial and Fungal are displayed in Table 4 and Figure 1. The results of the study indicated that control Antibiotics against bacteria and Fungi showed different inhibitory zones. Antibiotics activity of AM (10ug), CIP(25ug) ,CF(30ug) , PZ (75ug) and PC (100ug) against Staphylococcus aureus were 19, 26, 20, 21, 20mm ; E. coli 17, 28, 18, 20, 19mm;  Pseudomonas aeruginosa 18, 30, 17, 21, 18 mm; Klebsilla sp. 20, 33, 22, 23, 17 mm, and Candida albicans 21, 31, 20, 19, 22 mm respectively. The antimicrobial activity of the methanolic extracts of Psidium guajava compared to the selected antibiotics against selected microorganism Table 5 and Plate 1 showed that all antibiotics gave higher inhibition zones than the two extract concentrations. Yet, the activity of the two concentrations was closest to Amoxycillin activity, but much lower than the resistant Staphylococcus aureus and Escherichia coli.

The antimicrobial activity of the aqueous extracts of Psidium guajava against   selected microorganisms was less in activity compared to all the selected antibiotics Table 6 and Plate 2. This study showed that Ciprofloxacin (30µg) gave the highest inhibition zone among all antibiotics with the selected organisms 26, 28, 30 mm against Staphylococcus aureus, E. coli, Pseudomonas aeruginosa respectively. In other study¹⁵ Ciprofloxacin (25µg)   gave high diameter of inhibition zone which reached up 19, 23, 23mm against Staphylococcus aureus, E. coli, Pseudomonas aeruginosa respectively. The majority of the antibacterial activity in this study was found in the methanolic rather than the aqueous extracts, and the highest activity was found in the methanolic extracts from Psidium guajava.  Similar results were achieved in a previous study¹⁶. Results from this study provide evidence for the medicinal values of the tested plants. It was showed that the antimicrobial activity of the methanolic and aqueous extracts of Psidium guajava leaves achieved different diameters of  the bacterial growth inhibition zone against  Klebsiella sp and E. coli, while  a study¹⁶ has mentioned that, the extract of  Psidium guajava  leaves had no any activity against Klebsiella sp and E. coli.¹⁷ explained that  the methanol extract of  Psidium guajava leaves had an antibacterial activity with mean zones of inhibition of 12.3 mm, against S. aureus, while, in this study, high diameter of 17mm was achieved from methanol extract of  Psidium guajava leaves Table 5 and Plate 1. In this study, the results from water extract of Psidium guajava leaves against E. coli. Showed that the diameter of inhibition zone reached up 14mm Table 6 and Plate 2, these are similar result achieved by 18, who mentioned that, the antibacterial effects of water  extracts from P. guajava (guava)  leaves demonstrated mean exhibited zones of inhibition of 13.7mm  on E. coli.

Antioxidant activity   

Results showed are 88.4%, highest from standard, ascorbic acid 87.5% (Table 11 and Figure 2). These results revealed that the value of the Psidium guajava leaves extract was superior to the control (88.4%). Another study carried out by a previous study¹⁹.  Results showed that the value of antioxidant activity in the guava extract was 94.4% at a concentration of 100 μg/ml, and the guava dried fruit extracts exhibited weaker antioxidant effects than did the leaf extracts. A study estimate the antioxidants in Psidium guajava leaves extract, showed  a significant role of plant leaves as an antioxidant²⁰. Similar results obtained another study⁶ where the antioxidants reached 82% in the full concentration of the leaves extract.

CONCLUSION

The present study showed that Psidium guajava are rich sources of useful secondary metabolites, It is strongly recommended of using them for general medicinal purpose and specially for treat wounds and burns diseases. It is strongly recommended of using them for production of effective pharmaceutical compounds and can be used as natural products of antimicrobial to treat wounds and burns diseases instead of chemical drugs. It is noticeable that the leaves of Psidium guajava are very rich in antioxidant content and therefore are good sources and safe and economical.

CONFLICT OF INTEREST

"No conflict of interest associated with this work”.

REFERENCES

1. Mani A, Mishra R, Thomas G. Elucidation of diversity among Psidium species using morphological and SPAR methods. J Phytol 2011 3(8): 53-61.
2. Abdelrahim    SI,  Almagboul  AZ,   Omer MEA, Elegami,  Antimicrobial activity of Psidium guajava L Fitoterapia. J List Int J   Microbiol 2002; 73(7-8), 713-715.           
3. Ramírez JC, Cura CI, da Cruz Moreira et al. Analytical validation of quantitative real-time PCR methods for quantification of Trypanosoma cruzi DNA in blood samples from Chagas disease patients. The J Mol Diag 2015; 17(5), 605-615.
4. Ncube NS, Afolayan AJ, Okoh AI. Assessment techniques of antimicrobial properties of natural compounds of plant origin: current methods and future trends. African J biotech 2008; 7(12): 1797-1806.‏
5. Keservani RK, Kesharwani RK, Vyas N, Jain S, Raghuvanshi R, Sharma AK. Nutraceutical and functional food as future food: a review. Der Pharmacia Lettre 2010; 2(1), 106-116.
6. Gokhale SB, Kokate CK, Purohit AP, Shah BN, Seth AK. Pharmacognostic studies of the Lagenaria siceraria (Molina) Standley. Int J Pharm Tech Res 2007; 2(1), 121-124.‏
7. Ciulci I. Methodology   for the    analysis   of   vegetable    Chemical industries branch, Division of industrial operations". UNIDO Romania: 1994; 24, 26 and 67.        
8. Tanko Y, Abdelaziz MM, Adelaiye AB, Fatihu MY, Musa KY. Effects of hydromethanolic leave extract of Indigofera pulchra on blood glucose levels of normoglycemic and alloxan-induced diabetic Wistar rats. Int J App Res Nat Prod 2008; 1(4), 13-18.‏
9. Puntawong S, Okonogi S, Pringproa K. In vitro antibacterial activity of Psidium guajava leaf extracts against pathogenic bacteria in pigs. Chiang Mai Univ J Nat Sci 2012; 11, 127-34.
10. Mensor LL, Menezes FS, Leitão GG, Reis AS, Santos TCD, Coube CS, Leitão SG. Screening of Brazilian plant extracts for antioxidant activity by the use of DPPH free radical method. Phytotherapy research 2001; 15(2), 127-130.‏
11. Gitika, Kumar M. Antibacterial activity of Psidium guajava leaves extracts against some gram-positive and gram-negative bacteria. Europ J Pharm Med Res 2016;3(10), 261-266.
12. Kangogo KG, Kagira MJ, Maina N, Karanja MS. Qualitative phytochemical screening of camellia sinensis and psidium guajava leave extracts from Kericho and Baringo Counties. 2014; 5(3): 506-512.
13. Anbuselvi, S.,and Rebecca, J. Phytochemical biochemical antimicrobial activty of psidium guajava leaf extract. J Pharm Sci Res 2017; 9(12), 2431-2433.           
14. Abubakar EMM. (2009). Antibacterial activity of crude extracts of Euphorbia hirta against some bacteria  associated  with enteric infections. J Med Plants Res 2009; 3(7):498-505.
15. Siti NS, Kamaruzaman S. Antibacterial activity of Psidium guajava Methanol leaf extract against plant pathogenic bacteria in the genera Pectobacterium and Xanthomonas. Int J Appl Biol Pharmac Technol 2012; 3, 246-252.
16. Bhalodia NR, Shukla VJ. Antibacterial and antifungal activities from leaf extracts of Cassia fistula l.: An ethnomedicinal plant". J advanced pharm tech research 2011; 2(2), 104.
17. Rabe T, Van Staden J. Antibacterial activity of South African plants used for medicinal purposes. J ethnopharmacology 1997; 56(1), 81-87.‏
18. Biswas B, Rogers K, Fredrick M, Dwayne Borde VU, Pawar DP, Shelar SR, Apturkar Antimicrobial activity of some medicinal plants". Science Research Reporter;2013; 3(1): 33-37.‏
19. Adefuye A, Samie A, Ndip. In-vitro evaluation of the antimicrobial activity of extracts of Bridelia micrantha on selected bacterial pathogens. J Med Plant Res 2011; 5, 5116-5122.
20. Chen HY, Yen GC. Antioxidant activity and free radical-scavenging capacity of extracts from guava (Psidium guajava) leaves. Food chemistry 2007; 101(2): 686-694.‏
21. Mishra R., Tiwari P, Srivastava M, Singh CS, Ghoshal S. A Comprehensive Review On Psidium guajava Linn (Amaratafalam)". World 2007; 7, 8.‏

Table 1: R_f values of TLC solvent system for different extracts of Psidium guajava.

Table 2: Yields of Psidium guajava leaves extracts from methanolic and aqueous extracts.                                                                                                       

Mean values of the yield are presented as mean ± SEM, Values are statistically,   significant when p≤ 0.05.

Table 3: Phytochemical composition of the methanolic and aqueous leaves extracts of Psidium guajava. 

Absence (+), Presence (-)

. Table 4: Antimicrobial activity of standard antibiotics discs against tested bacterial and fungal.

AM=Amoxycillin, CIP= Ciprofloxacin, CF=cefazllin, PZ=Cefoperazone, PC=piperacillin

Table 5: Antimicrobial activity of the methanolic extracts of leaves of (Psidium guajava) and standard antibiotics discs against tested bacterial and fungal.     

Figure 1: Antimicrobial activities (inhibition zones mm.) of standard antibiotics discs against tested bacterial and fungal.

Table 6: Antimicrobial activity of the aqueous extract of leaves (Psidium guajava) and standard antibiotics discs against tested bacterial and fungal.

Table 7: Antioxidant activities of the selected extracts and L- ascorbic acid using the

(DPPH) free radical-scavenging assay.

Figure 2: Antioxidant activities of the selected extracts and L-ascorbic acid using the (DPPH) free radical-scavenging assay.

Plate 1: Inhibition zones observed with leaves methanolic extracts of Psidium guajava.

Plate 2: Inhibition zones observed with leaves aqueous extracts of Psidium guajava.