Authors
Nantenaina Tombozara1, Andrianambinina A. Razakarivony2, Zoarilala Rinah Razafindrakoto1, Rodrigue Keumoe3, Steven Njonte Wouamba4, Simeon Fogue Kouam4, Bruno Ndjakou Lenta4, David Ramanitrahasimbola1,5, Reine Dorothée Ramilison–Razafimahefa2, Dina Andriamahavola Rakotondramanana5, and Norbert Sewald6
Affiliations
1. Institut Malgache de Recherches Appliquées, Laboratoire de Pharmacognosie Appliquée, Antananarivo, Madagascar.
2. Université d’Antananarivo, Faculté des Sciences, Département de Chimie Organique, Laboratoire de Chimie Appliquée aux Substances Naturelles, Madagascar.
3. University of Yaoundé 1, Faculty of Sciences, Biochemistry Department, Antimicrobial and Biocontrol Agents Unit, Yaoundé, Cameroon.
4. University of Yaoundé 1, Higher Teachers’ Training College, Chemistry Department, Yaoundé, Cameroon.
5. Université d’Antananarivo, Faculté de Médecine, Département de Pharmacie, Antananarivo, Madagascar.
6. Bielefeld University, Faculty of Chemistry, Organic and Bioorganic Chemistry, Bielefeld, Germany.
Corresponding Author: Mr. Nantenaina Tombozara, Institut Malgache de Recherches Appliquées, Laboratoire de Pharmacognosie Appliquée, P.O. Box 3833, Antananarivo, Madagascar. Phone: +261341015902 ; Email: nzara89@gmail.com
Abstract
Background: Madagascar is known by the very high endemicity rate of its flora, this richness in floral biodiversity could be an inexhaustible resource of the bioactive molecules. This study aims to investigate seven extracts from leaves and barks of five Malagasy plants for antibacterial and antifungal activities according to their ethno-pharmacological use in local pharmacopeia to treat different diseases.
Materials and methods: Phytochemical screening of all extracts was undertaken using the common coloration and precipitation methods. Broth micro-dilution antimicrobial assay was used to assess their in vitro antimicrobial activity on 19 bacterial and 4 yeast strains.
Results: Phytochemical screening allowed to detect alkaloids, flavonoids, anthocyanins, leucoanthocyanins, terpenoids, steroids, unsaturated sterols, cardiac glycosides, phenolic compounds, tannins, quinones, polysaccharides, and saponins. The leaves extract of Schefflera bojeri exhibited the most potent inhibitory activity against Staphylococcus aureus amoxicillin-resistant strain (BAA917) with a minimum inhibitory concentration (MIC) of 125 µg/ml. Moreover, the stem-bark extract of this plant exerted the same MIC of 500 µg/ml on two strains of S. aureus (ATCC43300 and NR4674) and Pseudomonas aeruginosa (NMC952). The antibacterial activity of the leaves extract of Uapaca bojeri was active against the S. aureus (BAA917) amoxicillin-resistant strain (MIC = 250 µg/mL) and Streptococcus pneumoniae (ATCC49619, MIC = 500 µg/mL). The leaves extract of Crassula ovata showed a moderate MIC (500 µg/ml) against Salmonella enterica (NR4249) amoxicillin-resistant strain and Klebsiella pneumoniae (ATCC13883).
Conclusion: The phytochemical and antimicrobial screening pro-Gram allows us to confirm the therapeutic value of these medicinal plants which need further investigation of the isolation of the active compounds.
Key words: Antimicrobial activity, Crassula ovata, Schefflera bojeri, Uapaca bojeri, Malagasy pharmacopeia
1. Introduction
Madagascar is known by the very high endemicity rate of its flora, estimated to 80% of 12,000 to 14,000 species (Robinson, 2004). Until now, only 10% of them have been biologically investigated (Hudson et al., 2000) and the antimicrobial activity of the Malagasy endemic medicinal plants is poorly investigated (Hudson et al., 2000; Rasoanaivo et al., 2004; Rakotoniriana et al., 2010; Velomalala et al., 2013). The traditional medicine plays an important role in the Malagasy health system and could provide the opportunity to discover new chemical compounds possessing an interesting pharmacological profile and therapeutic uses. Plants could be an inexhaustible resource of the bioactive molecules.
Infectious diseases constitute the main concern of the public health of tropical countries. Irrational use of antibiotics nowadays resulted in the development of resistant strains (Khatri et al., 2016). Due to the emergence of this phenomenon, there is an urgent need for new antibiotics. Infectious diseases caused by antibiotic-resistant germs have escalated world-wide, rising the patient morbidity, mortality, and health care costs. In Madagascar, infectious complications are the second cause of morbidity and mortality (Ranivoharisoa et al., 2015). For many reasons, most of the Malagasy people rely on traditional medicine for the care of their health. The active principles of many phytodrugs are secondary metabolites. The remarkable contribution of plants to the drug discovery was possible because of the large number of the phytochemical and biological studies all over the world. This study was conducted to validate some traditional uses of plants in Malagasy pharmacopeia, particularly for antibacterial and antifungal activities in order to scientifically valorize the potentiality of the flora biodiversity of Madagascar.
2. Materials and methods
2.1 Collect and plant materials
Five plants (Table 1) are selected from a Malagasy pharmacopeia book according to them traditional uses. A short interview was undertaken with seven to ten respondents concerning the ethno-medicinal uses and preparation of these plants during their collect. Aerial parts and barks of S. bojeri, U. bojeri and V. secundiflorum were collected in the Tapia hill of Imamo at 70 km in the western part of Antananarivo. Aerial parts of S. jamaicensis were collected at Mangamila, at about 50 km in the north of Antananarivo and leaves of C. ovata were harvested in Antananarivo. All specimens were collected in January 2017 and identified by Mr. Rakotonirina Benja, the botanist of Institut Malgache de Recherches Appliquées (IMRA) and voucher specimens have been deposited at the IMRA Botany Department respectively under TN-020/LPA, TN-021/LPA, TN-022/LPA, TN-010/LPA and CON02/LPA identities. Leaves and barks of S. bojeri and U. bojeri were separated and all collected specimen were dried in a shady aerated place before grinding.
2.2 Extraction preparation
Plant powders (25 g) were extracted by maceration with 250 mL of ethanol (96%) during 24 hours at room temperature with intermittent shaking. The extraction process was repeated 3 times and all the alcoholic solutions were combined and evaporated to dryness at 40 °C bath temperature under reduced pressure.
2.3 Phytochemical screening
The classes of secondary metabolites were detected by the classical methods for this purpose described in our previous work (Tombozara et al., 2017). The protocols were based on the formation of colored soluble or precipitated compounds by the specific reactive reagent used.
2.4 Antibacterial and anti-fungal assays
2.4.1 Microorganisms and culture condition
The antimicrobial activity was performed against nineteen bacterial strains (Table 2) and against four yeast strains (Table 3) provided by Be resource (ATCC) and the Hôpital Central de Yaoundé for clinical isolates. All the microorganism strains were maintained in culture on Mueller Hinton Agar (MHA) for bacterial and Sabouraud Dextrose Agar (SDA) supplemented with chloramphenicol for yeast according to the manufacturer’s instructions and growth at 37 °C for 24 h and 48 h respectively.
2.4.2 Inoculums’ preparation
The inoculums were prepared from a 1 day and 2 days old culture of bacteria and yeast, respectively, by scraping the colonies and putting them into a sterile test tube containing 5 mL of autoclaved 0.85% aqueous sodium chloride solution. The turbidity of the suspension was compared to a 0.5 Mc Farland standard solution corresponding to 1.5 × 108 CFU/mL for bacteria and 2.5 × 106 CFU/mL for yeast.
2.4.3 Preparation of plant extracts stock solution and antibiotics
The plant crude extracts stock solutions were prepared at 2 mg/mL in dimethylsulfoxid (DMSO) then diluted in sterile distillated water to reduce the DMSO rate in the tested solution to 2%. The reference products were amoxicillin (Sigma-Aldrich) for bacteria and fluconazol (Sigma-Aldrich) for yeast, both prepared at a concentration of 512 µg/mL in sterile distilled water. All the prepared stock solutions were stored at 4 °C for later use.
2.4.4 Antimicrobial assay
The broth micro-dilution assay was used to determine the Minimum Inhibitory Concentration (MIC) of the tested samples against bacterial (Table 2) and yeast (Table 3) strains in the 96 well-microtiter plate according to the Clinical and Laboratory Standards Institute (CLSI) guideline protocols M07-A9 (CLSI, 2012a) and M27-S4 (CLSI, 2012b), respectively. Mueller Hinton Broth (MHB) and Sabouraud Dextrose Broth (SDB) supplemented with chloramphenicol were used as culture media for bacteria and yeast susceptibility severally. Briefly, the microtiter plates for bacteria and yeast were prepared by dispensing 50 µL of MHB and SDB. Later, 50 µL aliquots of stock solution of plants extracts, or antibiotic / antifungal controls were added onto the first well followed by two-fold serial dilution after mixing thoroughly. 50 µL of the inoculums concentrated at 106 CFU/mL for bacteria and 105 CFU/mL for yeast were introduced in each well except for the blank containing only culture medium and representing the sterility control. The wells containing the inoculums without test substances constituted the negative control and the positive controls were amoxicillin for bacteria and fluconazol for yeast. The concentration of plant extract in the plate ranged from 3.9 to 500 µg/mL and from 1 to 128 µg/mL for reference drugs. The microtiter plates were incubated at 37 °C for 24 h or 48 h depending to the microorganism. After the incubation time the lowest concentration of test samples inhibiting the visible growth of the bacteria or yeast as observed by the turbidity of the medium was recorded as the MIC. Each experiment was performed in triplicate.
3. Results and discussion
3.1. Ethnomedicinal uses
The ethnomedicinal uses of the selected plants were reported in the table 1. Schefflera bojeri, Uapaca bojeri and Vaccinium secundiflorum are shrubs which essentially grow on four dislocated ecological zones of the highlands of Madagascar and constitute some veritable hills of Tapia including the hill of Imamo at the Itasy region, the hill of Manandona and in Itremo at the Imoromania region and the hill of Isalo in Horombe region (Kull et al., 1999). These endemic plants are locally used to serve as the main host for the Malagasy silk worms breed called landibe (Borocera cajani) for the fabrics production (Verheggen et al., 2013). Several phytocompounds belonging to the phenolics, organic acids, vitamins, tannins have been quantified by Tombozara et al. (2020) and Razafindrakoto et al. (2020) respectively from the aerial parts of V. secundiflorum and U. bojeri. Their studies demonstrated the antidiabetic, analgesic and anti-inflammatory of these plants. Crassula ovata is an ornamental plant evergreen originated from South Africa, its common name was given due to its shape like baobab (Tombozara et al., 2017). Its leaves were mainly used by the local population to treat hypertension by chewing 7 leaves by swallowing the juice and discarding the rest during the occurrence of headache or dizziness and also during the stomachache. Stachytarpheta jamaicensis is an herbaceous annual plant originated from Caribbean; its common name was given from its long ear like a rat-tail (Duret et al., 1976). Its traditional uses are similar to that of others country (Liew et Yong, 2016).
3.2. Extraction and phytochemical screening
The yields of the extraction were reported in the table 4. Maceration in ethanol (96%) produced high-level yields due to the similarity of polarity of most of the plant components. Comparing into our previous work (Tombozara et al., 2017), the extraction yield of C. ovata decreased from 18.4% to 4.45% and the relative proportion of phenolic compounds, tannins and polysaccharide also decreased showing that the period of collection might affect the biosynthesis of secondary metabolites in this plant. The detected classes of secondary metabolites from these plants included alkaloids, flavonoids, anthocyanins, leucoanthocyanins, terpenoids, steroids, unsaturated sterols, cardiac glycosides, phenolic compounds, tannins, quinones, polysaccharides and saponins (Table 3) according to the intensity of color or precipitates. Low amounts of alkaloids were detected in S. bojeri and V. secundiflorum showing that these species might be sources of alkaloids. Flavonoids and saponins were detected in all extracts excepted for leaves of C. ovata. Flavonoids have been reported to possess many useful properties, including anti-inflammatory, estrogenic, enzyme inhibition, antimicrobial, anti-allergic, antioxidant, vascular, antidiabetic and cytotoxic antitumor activity (Havesteen, 1990; Harborne and Williams, 2000; Kasali et al., 2016). Leucoanthocyanins and anthocyanins were detected as a major phyto-constituent of all crude ethanol extracts. These classes of secondary metabolite are well-known in their contribution in antioxidant, antimicrobial and antidiabetic activity (Mccune and John, 2007; Grace et al., 2009; Wang et al., 2014), that may contribute to some plant parts use to the diabetes and infectious diseases treatment. Beside them, tannins, one of the considerable phyto-compounds in nutritional, health and medicine fields due to their antioxidant, antimicrobial and anti-inflammatory properties (Santos-Buelga and Scalbert, 2000), were also detected in all extract. On the other hand, lactonic steroids were absent for all species. Lactonic compounds are known for their toxicity (Mikkola et al., 2000; Aguedo et al., 2002) but some further toxicity studies will be necessary to confirm these hypotheses. Traces of phenolic compounds were detected only in leaves of C. ovata, aerial parts of S. jamaicensis and stem-barks of U. bojeri. The chemical composition of S. jamaicensis varied also according to the collecting zone. In Madagascar, it does not contain any alkaloid (Table 5). The results of this work are in perfect agreement with those by Duret et al. (1976). Ruma and Zipagang (2015) report a similar phytochemical result from the Philippines. Nevertheless, alkaloids were detected in specimen harvested in India and Nigeria (Okwu and Offiong, 2009; 2010; Ramakrishnan and Sivaranjani, 2013; Pandian et al., 2013; Sivaranjani et al., 2013; Iroka et al., 2015). The edaphic conditions could be at the origin of these differences. Remarkably, we did not detect any steroid compounds in samples containing terpenoids like leaves and stem barks of S. bojeri and in leaves of U. bojeri or unsaturated sterols (S. bojeri, U. bojeri and V. secundiflorum). Vice versa, in the other extracts containing steroids, terpenoids were absent. However, the most of the reported phytochemicals quantified by Tombozara et al. (2020) and Razafindrakoto et al. (2020) have been detected during this study showing the effectiveness of the used methods.
3.2 Antimicrobial activities
Antibacterial and antifungal activities of the ethanol extract of the samples were reported in the tables 2 and 4 respectively. The ethanol extract of S. bojeri and U. bojeri leaves showed the lowest MIC, 125 μg/mL and 250 μg/mL, respectively, against S. aureus BAA917, which was an amoxicillin-resistant strain. The stem bark of S. bojeri also exhibited a moderate antibacterial activity (MIC = 500 μg/mL) against two strains f S. aureus (ATCC43300 and NR4674) and P. aeruginosa (NMC952). The ethanol extract of U. bojeri leaves was the only extract active against S. pneumoniae ATCC49619 with a moderate activity (MIC = 500 μg/mL). Interestingly, the crude extract of C. ovata leaves exhibited a moderate inhibitory effect against two Gram-negative bacterial strains including S. enterica NR4249, a highly amoxicillin-resistant strain, and K. pneumoniae ATCC13883. Crude ethanol extracts of S. jamaicensis and V. secundiflorum aerial parts and the crude ethanol extract of U. bojeri stem bark did not show any antibacterial activity. All the crude extracts did not exhibit any activity against fungal strains. This does not mean that they were inactive but active compounds might be present in low quantity so they are hidden or interfered by other secondary metabolites.
S. aureus is an opportunist Gram-positive bacterium and considered as a major responsible of skin, soft tissue, respiratory, bone, joint, and endovascular disorders (Lowy, 1998; Klevens et al., 2007; Venkanna et al., 2013; Stevens et al., 2014). The crude extracts of the leaves of U. bojeri and S. bojeri showed a very good inhibition against S. aureus, which were in agreement with their traditional uses to treat wounds and abscesses and accelerate their healing. The major constituents of the three active extracts on S. aureus strains were terpenoids, saponins, leucoanthocyanins and tannins, or acid tannins, as astringent, which are known for their antibacterial and antioxidant activities especially to inhibit S. aureus (Rivière et al., 2009; Eahamban and Antonisamy, 2012; Joseph et al., 2015; Tintino et al., 2017). Terpenoids and saponins are also known for their anti-inflammatory, hepato-protective, analgesic, antimicrobial, antimitotic, virostatic, immuno-modulatory and tonic effects (Eahamban and Antonisamy, 2012). Therefore, they could be responsible for the activity of these plants when they were used in the traditional treatments. S. pneumoniae, a Gram-positive bacterium, is an important resistant germ pathogen in human. It causes several diseases including pneumonia, meningitis, sinusitis and otitis media, especially in children (Kim et al., 2016; Lee et al., 2017; Andrade et al., 2017). Combined with S. aureus, it causes a contagious infection as pyomyositis (Korusawa et al., 2017). Several phytomedicines have already been used to treat infectious diseases, especially caused by S. aureus and S. pneumonia (Kuete, 2010; Igwe and Johnbull, 2013; Shalayel et al., 2016). Leaves of U. bojeri could be considered as an alternative of one them because they were active against the both of these strains. P. aeruginosa is a Gram-negative bacterium which causes acute or chronic infections in animals and humans, including respiratory infections in cystic fibrosis, nosocomial infections, bacteremia, pneumonia, urinary tract infections, burn infections, ocular infections, malignant or simple external otitis, folliculitis and other skin infections, chronic otitis media, osteomyelitis, septic arthritis, endocarditis, brain abscesses, meningitis, decubitus ulcer and green nails (Deretic, 2000). The stem bark of S. bojeri might be a preferred phytomedicine to cure some of these diseases. The crude extract of C. ovata moderately inhibited both the S. enterica NR4249 and K. pneumoniae ATCC13883. S. enterica, a Gram-negative bacterium, causes diseases such as diarrhea, fever and abdominal pain (Rodríguez et al., 2017) mainly due to contaminated food and water. Rare cases of Salmonella enterica subsp. arizonae infections were reported after direct or indirect contact with reptile or consumption of snake products (Nishioka et al., 2017). K. pneumoniae is an opportunist Gram-negative bacterium which mainly causes urinary tract, respiratory tract or bloodstream infections, skin, nose, throat diseases and liver abscess (Holt et al., 2015; Fazili et al., 2016). Almost all antibiotics used to inhibit K. pneumoniae are nitrogenized compounds including ampicillin, amoxicillin, piperacillin, ceftazidime, cefotaxime, cefepime, meropenem, ertapenem and levofloxacin (Pichler et al., 2017). Yet C. ovata was devoid of nitrogen compounds. This means that the extract may contain non-alkaloid antibiotics which may be leucoanthocyanin or tannin compounds, the major constituents of this plant. Muiruri and Mwangi (2016) tested leave and stem extracts on five strains where the only Gram-negative strain, E. coli, was inhibited with a fairly high concentration. Therefore C. ovata leaves contain secondary metabolites that only inhibit Gram-negative strains.
4. Conclusion
This study was a preliminary evaluation of antimicrobial activity of these plants. It indicated that four plant extracts have the potentiality to generate novel active metabolites; three crude extracts displayed activity against resistant strains, which could result in the discovery of novel antimicrobial agents. The plants demonstrating broad spectra of activity may help to discover new chemical classes of antibiotics that could serve as selective agents for the maintenance of animal or human health and provide biochemical tools for the study of infectious diseases. The micro-dilution tests have been carried out at low concentrations (≤ 500 μg/mL) to yield only very relevant results, which did not allow concluding that the other extracts were non-active or did not contain active molecules but may be by increasing the concentration, activity could turn out. This works reports also the first investigation of the three endemic plants for their antimicrobial properties. The different pathologic settings where these plants are being used in traditional and folk medicines as well as in pharmacopoeia confirm the results obtained. Further studies are needed including the isolation of the active compounds for the confirmation of the antimicrobial properties to provide safer standardized phytomedicines.
Abbreviations
CFU: Colony forming unit
CLSI: Clinical and Laboratory Standards Institute
DMSO: Dimethylsulfoxide
IMRA: Institut Malgache de Recherches Appliquées
MHA: Mueller Hinton Agar
MHB: Mueller Hinton Broth
MIC: Minimum Inhibitory Concentration
SDA: Sabouraud Dextrose Agar
SDB: Sabouraud Dextrose Broth
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
NT drafted the manuscript. NT, ZRR, DR and DAR carried out plant collection and ethno-medicinal investigation. NT, ZRR and AAR performed the plant extraction and the phytochemical screening. RK and SNW performed the in vitro antimicrobial assays under the supervision of SFK and BNL. RK, DR, RDRR, DAR and NS reviewed and edited the manuscript. All authors read and approved the final manuscript.
Funding
This work was supported by the German Academic Exchange Service (DAAD) through Yaoundé – Bielefeld Bilateral Graduate School Natural Products with Antiparasite and Antibacterial Activity (YaBiNaPA) project.
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Table 1: Traditional uses of selected plants
| Family and binomial name | Common name | State | Used organ | Preparation mode | Traditional uses | |
| ARALIACEAE Schefflera bojeri (Seem.) R. Vig. | Sakaizatapia | Endemic | Leaf | Infusion | Headache and dizziness, tonic cardiac | |
| Chewed and deposited | Wounds, sores, abscesses | |||||
| Stem bark | Infusion or decoction | Diabetes | ||||
| CRASSULACEAE Crassula ovata (Mill.) Druce | Baobabakely or Reozo | Introduced (ornamental) | Leaf | Chewed and swallowed the juice | Headache and dizziness, tonic cardiac, stomachache, diarrhoea, liver and diabetes | |
| EUPHORBIACEA Uapaca bojeri Bail. | Tapia | Endemic | Leaf | Infusion | Headache and dizziness, tonic cardiac | |
| Chewed and deposited | Wounds, abscesses, swelling and inflammation | |||||
| Decoction | Diarrhoea, stomach affection | |||||
| Stem bark | Infusion | Diabetes | ||||
| Decoction | Diarrhoea | |||||
| Rape fruit | No preparation | Used as food complementary | ||||
| ERICACEAE Vaccinium secundiflorum Hook. | Voatsitakajaza or Kitondra | Endemic | Aerial part | Infusion | Diabetes | |
| Leaf | Decoction | Diarrhoea, stomach affection | ||||
| Rape fruit | No preparation | Used as food complementary | ||||
| VERBENACEAE Stachytarpheta jamaicensis L. (Vahl) | Rambomboalavo | Introduced | Aerial part | Infusion | Diabetes | |
| Decoction | Stomachache, Diarrhoea | |||||
| Whole plant | Freshly grinded and macerated in water | Malaria, diabetes, cold, itch, wounds, sores | ||||
Table 2: MIC (μg/ml) of plant extracts against bacterial strains
| Bacterial strain | C. ovata | S. bojeri | S. jamaicensis | U. bojeri | V. secundiflorum | Controls | ||
| Leaves | Leaves | Stem-barks | Aerial parts | Leaves | Stem-barks | Aerial parts | Amoxicillin | |
| Streptococcus pneumoniae ATCC49619 | >500 | >500 | >500 | >500 | 500 | >500 | >500 | 8 |
| Staphylococcus aureus BAA917 | >500 | 125 | >500 | >500 | 250 | >500 | >500 | >128 |
| Staphylococcus aureus ATCC43300 | >500 | >500 | 500 | >500 | >500 | >500 | >500 | 16 |
| Staphylococcus aureus NR45003 | >500 | >500 | >500 | >500 | >500 | >500 | >500 | 1 |
| Staphylococcus aureus NR46003 | >500 | >500 | >500 | >500 | >500 | >500 | >500 | >128 |
| Staphylococcus aureus CP7625 | >500 | >500 | >500 | >500 | >500 | >500 | >500 | 1 |
| Staphylococcus aureus NR4674 | >500 | >500 | 500 | >500 | >500 | >500 | >500 | 4 |
| Shigella flexineri NR518 | >500 | >500 | >500 | >500 | >500 | >500 | >500 | >128 |
| Salmonella enterica NR4249 | 500 | >500 | >500 | >500 | >500 | >500 | >500 | >128 |
| Salmonella enterica NR4311 | >500 | >500 | >500 | >500 | >500 | >500 | >500 | 32 |
| Salmonella enterica NR13555 | >500 | >500 | >500 | >500 | >500 | >500 | >500 | 8 |
| Pseudomonas aeruginosa NMC952 | >500 | >500 | 500 | >500 | >500 | >500 | >500 | 1 |
| Klebsiella pneumoniae ATCC13883 | 500 | >500 | >500 | >500 | >500 | >500 | >500 | 4 |
| Klebsiella pneumoniae ATCC70613 | >500 | >500 | >500 | >500 | >500 | >500 | >500 | 1 |
| Klebsiella pneumoniae NR41916 | >500 | >500 | >500 | >500 | >500 | >500 | >500 | 4 |
| Escherichia coli ATCC25922 | >500 | >500 | >500 | >500 | >500 | >500 | >500 | 2 |
| Escherichia coli ATCC35218 | >500 | >500 | >500 | >500 | >500 | >500 | >500 | >128 |
| Enterococcus fecalis ATCC51219 | >500 | >500 | >500 | >500 | >500 | >500 | >500 | >128 |
| Hemophyllus influenza ATCC49247 | >500 | >500 | >500 | >500 | >500 | >500 | >500 | 1 |
| > 500: low activity; ≤ 500: average activity; ≤ 250: good activity and ≤ 125: very good activity |
Table 3: MIC (μg/ml) of plant extracts against fungal strains
| Fungal strain | C. ovata | S. bojeri | S. jamaicensis | U. bojeri | V. secundiflorum | Controls | ||
| Leaves | Leaves | Stem-barks | Aerial parts | Leaves | Stem-barks | Aerial parts | Fluconazol | |
| Candida parapsilosis | >500 | >500 | >500 | >500 | >500 | >500 | >500 | 4 |
| Candida albicans | >500 | >500 | >500 | >500 | >500 | >500 | >500 | 8 |
| Candida Krusei | >500 | >500 | >500 | >500 | >500 | >500 | >500 | 2 |
| Cryptococcus neoformans | >500 | >500 | >500 | >500 | >500 | >500 | >500 | 1 |
| > 500: low activity; ≤ 500: average activity; ≤ 250: good activity and ≤ 125: very good activity |
Table 4: Yield extraction of each plant powder
| Plant specie | C. ovata | S. bojeri | S. jamaicensis | U. bojeri | V. secundiflorum | ||
| Leaves | Leaves | Stem-barks | Aerial parts | Leaves | Stem-barks | Aerial parts | |
| Mass (g) | 1.113 | 2.625 | 1.91 | 2.987 | 2.290 | 1.635 | 2.825 |
| Yield (%)* | 4.45 | 10.50 | 7.64 | 11.95 | 9.16 | 6.54 | 11.30 |
| * relative proportion to plant powder |
Table 5: Classes of secondary metabolites of each selected plant
| Class of secondary metabolites | C. ovata | S. bojeri | S. jamaicensis | U. bojeri | V. secundiflorum | ||
| Leaves | Leaves | Stem-barks | Aerial parts | Leaves | Stem-barks | Aerial parts | |
| Alkaloids | – | + | ++ | – | – | – | + |
| Flavonoids | – | +++ | +++ | + | +++ | +++ | +++ |
| Anthocyanins | + | + | + | + | +++ | +++ | +++ |
| Leucoanthocyanins | ++ | +++ | +++ | +++ | +++ | +++ | +++ |
| Terpenoids | – | +++ | +++ | – | +++ | – | – |
| Steroids | + | – | – | ++ | – | +++ | +++ |
| Lactonic steroids | – | – | – | – | – | – | – |
| Unsaturated sterols | – | +++ | +++ | – | + | ++ | +++ |
| Cardiac glycosides | – | ++ | ++ | + | – | – | – |
| Phenolic compounds | ++ | – | – | – | – | + | – |
| Tannins | ++ | +++ | +++ | + | +++ | +++ | +++ |
| Quinones | – | – | – | – | +++ | ++ | ++ |
| Polysaccharides | ++ | +++ | +++ | ++ | ++ | – | + |
| Saponins | – | ++ | ++ | + | ++ | ++ | ++ |
| +++: High content of phyto-compound ++: Average content of phyto-compound +: Low content of phyto-compound – : Absence of phyto-compound |