Synergistic Antimicrobial Activities and Phytochemical Analysis of Leaf Extracts from Sarcocephalus latifolius, Morinda lucida and Anogeissus leiocarpus

Received 23 Feb, 2024

Accepted 14 May, 2024

Published 31 Dec, 2024

Background and Objective: Morinda lucida (ML), Anogeissus leiocarpus (AL) and Sarcocephalus latifolius (SL) have long been valued in traditional medicine for their therapeutic properties. In Nigeria, a decoction of their leaves is commonly utilized to address various health issues, including lymphatic filariasis, often complicated by wounds or sores. The present study aimed to assess the phytochemical profile and synergistic antimicrobial activity of leaf extracts from Morinda lucida, Anogeissus leiocarpus and Sarcocephalus latifolius. Materials and Methods: The extracts of ML, AL, SL and a combination thereof were prepared via decoction and subjected to phytochemical analysis and high-performance liquid chromatography. The extracts were further evaluated for their antioxidant potential against DPPH and antimicrobial effects against a range of pathogens. Results: Phytochemical analysis unveiled the presence of bioactive compounds like saponins, terpenes, sterols, phenolics and tannins. The high-performance liquid chromatography elucidated the specific chemical profiles of each extract, with ML featuring prominent peaks of betulinic acid, chlorogenic acid, caffeic acid, rutin and ferulic acid. The AL showcased major peaks of gallic acid, chlorogenic acid and caffeic acid, while SL exhibited significant peaks of betulinic acid, gallic acid, caffeic acid and rutin. The combination extract displayed peaks corresponding to these compounds. Conclusion: Remarkably, AL demonstrated the highest antimicrobial and antioxidant activities, followed by the combination extract. These findings offer promising insights into the therapeutic potential of combined herbal therapy for lymphatic filariasis, warranting further investigation.

INTRODUCTION

Medicinal plants have been used as sources of drugs by mankind for several years. In fact, ancient man was totally dependent on plants for his needs of treatment, prevention and other forms of medicaments, thus utilizing plants as drugs for millennia¹. For the past 3000 years, a large number of plants are used in health care practices, such as Traditional Medicine in China, India and Africa, most of which contain therapeutic values which have been ascertained as such by the Western standards². Furthermore, several other plants have been employed for centuries by several cultures which are less likely to be proven by Western standards. Medicinal plants however remain a major source of novel drug discovery and development and have contributed greatly to human health. Nature’s pharmacy has bestowed humans with an endless supply of plants enriched with various secondary metabolites for man to harness. Plants have provided an endless array of chemicals for man since the beginning of man’s existence. Several oxidation reactions take place in the biological system resulting in oxidative stress, one of such is the univalent reduction of oxygen which results in the conversion of a certain percentage of inhaled oxygen into reactive species or free radicals called reactive oxygen species (ROS)³. Antioxidants are also called free radical scavengers and are compounds whose presence even at low concentrations possess the ability to prevent oxidation of cell constituents like lipid, carbohydrate, DNA and protein; they act via a number of processes such as by preventing the formation of free radicals through preventing phagocyte activation; through binding of transition metal ions and prevention of hydroxyl formation and via repair of damage as in tocopherol which repairs peroxyl radicals thereby halting lipid peroxidation and preventing decomposition of lipid hydroperoxide⁴.

A decoction made from the leaves of Morinda lucida, Anogeissus leiocarpus and Sarcocephalus latifolius is commonly used in Nigeria as a traditional remedy for treating lymphatic filariasis, even in cases complicated by wounds or sores^5,6. Morinda lucida, scientifically known as Benth and belonging to the Rubiaceae family, is referred to as Brimstone tree in English, Erhan Ikpanro in Benin, Huka or Eze ogu in Igbo and Oruwo in Yoruba⁷. This evergreen tree has a crooked or gnarled appearance, with leaves that are opposite, simple and entire⁸. The bark ranges from smooth to roughly scaly, while the wood is yellow in color^9,10. Morinda lucida has a rich history of safe consumption and has been reported to possess trypanocidal and antimalarial activities¹¹.

Anogeissus leiocarpus, belonging to the Combretaceae family, is known by various vernacular names such as African birch, Bambara, Anogeissus and chewstick¹². This graceful tree is native to the savannahs of tropical Africa and is characterized by its slow growth and grey to mottled pale and dark brown bark¹³. It has alternate to nearly opposite leaves covered in dense silky hair when young. Anogeissus leiocarpus is utilized in various traditional medicinal practices across Africa, including Nigeria, where it is used for treating coughs and maintaining dental hygiene¹³.

Sarcocephalus latifolius, a member of the Rubiaceae family, is known by names such as African Peach, Pin cushion tree and Guinea peach⁸. This savannah tree or shrub can grow up to 12 meters high and is characterized by its twisted bole and spreadingly open crown¹². The fruits of Sarcocephalus latifolius are fused together into a fleshy mass and the seeds are embedded in pinkish flesh with a strawberry scent. This plant is widely distributed throughout Africa and Asia and is reputedly used for treating malaria and other ailments¹⁴.

The present study aimed to assess the phytochemical profile and synergistic antimicrobial activity of leaf extracts from Morinda lucida, Anogeissus leiocarpus and Sarcocephalus latifolius. These plants have a long history of traditional use in managing various diseases and their combined use reflects the rich heritage of herbal medicine in Nigeria and other regions of Africa.

MATERIALS AND METHODS

Collection and identification of plant sample: Fresh leaves of Morinda lucida Benth, Anogeissus leiocarpus (D.C) Guill and Perr and Sarcocephalus latifolius J E Smith (E A Bruce) were harvested from the NIPRD garden and authenticated by Mr. Akeem Lateef, an expert taxonomist, at the herbarium of the National Institute for Pharmaceutical Research and Development in Abuja, Nigeria. This study was carried out from February, 2019 to July, 2020. Voucher specimens with the numbers NIPRD/H/7038, NIPRD/H/7039 and NIPRD/H/7040 were deposited for reference. The plant materials were then air-dried at room temperature for two weeks and subsequently ground into a fine powder using an electric grinder.

Preparation of plant extracts: The air-dried leaves were chopped into smaller pieces and weighed, with each plant material weighing 300 g for Morinda lucida, Anogeissus leiocarpus and Sarcocephalus latifolius. Additionally, a combination therapy consisting of 100 g each of Morinda lucida, Anogeissus leiocarpus and Sarcocephalus latifolius was prepared. The extraction process involved boiling the chopped leaves with distilled water for 10-15 min, using 750 mL of water, followed by allowing the mixture to stand overnight for 24 hrs. Afterward, the extracts were filtered using a funnel blocked with cotton wool. The resulting filtrates were then dried over a water bath to obtain dark brown extracts, with percentage yields of 3.3, 4.9 and 4.8% w/w, respectively.

Phytochemical analysis: Phytochemical analysis was conducted on the extracts for secondary metabolites and using standard methods of Wakawa et al.¹⁵.

High performance liquid chromatography analysis: The analysis of bioactive constituents in the extract was conducted using high-performance liquid chromatography (HPLC) with a UV diode array detector (UV-DAD). The HPLC setup included an Ultra-Fast LC-20AB equipped with a SIL-20AC auto-sampler, DGU-20A3 degasser, SPD-M20A UV-diode array detector, column oven CTO-20AC, system controller CBM-20Alite and Windows LC solution software, all from Shimadzu Corporation in Kyoto, Japan. The column used was a 5μm VP-ODS C18 with dimensions of 4.6×150 mm.

Chromatographic conditions were as follows: The mobile phase consisted of 0.2% v/v formic acid and acetonitrile in a ratio of 20:80; the mode was isocratic; the flow rate was set at 0.6 mL/min; 10 μL of a 100 mg/mL solution of the extract in water was injected; detection was performed at UV 254 nm. The HPLC operating conditions were programmed to maintain solvent B at 20% and the column oven temperature was set to 40°C. The total run time for the analysis was 30 min. For the identification of phytoconstituents, flavonoids and phenolic acid standards such as apigenin, rutin, quercetin, caffeic acid and ferulic acid were used, with their retention times compared under similar experimental conditions¹⁶.

Antimicrobial and antioxidant evaluations
Test organisms: Pure clinical isolates of Bacillus subtilis, Klebsiella pneumoniae, collected and biochemically confirmed from Diagnostic Laboratory of NIPRD clinic and American Type cultures of Escherichia coli (ATCC25952), Staphylococcus aureus (ATCC25923), Pseudomonas aeruginosa (ATCC 27853), Salmonella paratyphi (ATCC 9150), Mycobacterium bovis (27290) and Mycobacterium smegmatis (607) were used in this study.

Preparation of inoculum: A loopful of the respective test organisms (S. aureus, E. coli, P. aeruginosa, B. subtilis, K. pneumoniae and S. paratyphi) was aseptically transferred from the agar slants to 5 mL of nutrient broth and then placed in an incubator at 37°C. After 24 hrs of incubation, the cultures were diluted with normal saline to achieve turbidity equivalent to a 0.5 McFarland standard (106 CFU/mL) as described by Adamu et al.¹⁷.

The 50 microliters (50 μL) of each freshly thawed stock test organism (M. bovis and M. smegmatis) was inoculated into 50 mL of sterile Middlebrook 7H9/ADC media and then incubated at 37ºC with shaking for 5-7 days. The actively growing cultures of M. bovis and M. smegmatis were adjusted to an optical density between 0.2-0.3 at a wavelength of 650 nm using a Jenway 6405 UV-Visible spectrophotometer.

Antimicrobial activity of samples: Concentrations of each sample (100 mg/mL) were prepared and tested against the test organisms using the Agar well diffusion method. One hundred microliters (100 μL) of each standardized microorganism suspension was evenly spread on sterile molten Mueller Hinton agar in Petri dishes, which were then allowed to solidify. Aseptically, holes were bored using a sterile cork borer (6 mm) and the bottoms of the holes were sealed with a drop of Mueller Hinton agar. Then, 100 μL of each sample concentration were dispensed into the labeled wells. The plates were left to dry in a biosafety cabinet for approximately 2 hrs to allow the samples to diffuse. Subsequently, they were incubated at 37°C for 24-48 hrs. After incubation, antimicrobial activity was evaluated by measuring the zone of inhibition around each well and the average of the readings from duplicate plates was calculated¹⁸.

Antimicrobial activity: The antimicrobial test of the samples was carried out using the broth micro-dilution method in 96-well microtiter plates to determine the minimum inhibitory concentration (MIC). Each sample (500 mg) was dissolved in 5 mL of sterile water to obtain a stock concentration of 100 mg/mL, which was then serially diluted across the microtiter plate in two-fold dilutions. Duplicate assays were performed for each extract concentration. Mueller Hinton broth (50 μL) was dispensed into sterile wells of the microtiter plate from rows 1 to 12. Subsequently, 50 μL of the 100 mg/mL concentration of the samples was transferred into well 1 of the plate in duplicate. The contents of well 1 were then mixed thoroughly and 50 μL was transferred to well 2 and this process was repeated sequentially up to well 11, where 50 μL was discarded. Each of the wells (1-12) was inoculated with 50 μL of diluted organisms and incubated for 24 hrs at 37°C. After the dilution procedure and incubation, the final testing concentrations ranged from 25 to 0.0977 mg/mL. Following incubation, 25 μL of tetrazolium salt dye was added to all the wells and the plate was reincubated for 1 hrs. The wells were then observed for the absence or presence of microbial growth by examining the color change in the wells. The MIC was defined as the lowest concentration of the drug/extract that prevented the tetrazolium dye from changing color to pink. A colorless well indicated no microbial growth, while a pink color indicated the occurrence of growth^19,20.

Antioxidant activity
Free radical scavenging assays: The 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) assay is a widely used method to assess the radical scavenging capacity of antioxidant compounds. This assay relies on the reduction of DPPH, a stable free radical, in an alcohol solution when exposed to hydrogen-donating species, such as antioxidant compounds. This reduction results in the formation of the non-radical form of DPPH, known as DPPH-H, leading to a color change from purple to yellow, which can be quantified using a spectrophotometer. The disappearance of the purple color is measured at 517 nm. For the assay, a reaction mixture was prepared consisting of 1.0 mL of 0.3 mM DPPH in methanol, 1.0 mL of various concentrations of the extract (250, 125, 62.5, 31.25 and 15.625 μg/mL) and 1.0 mL of methanol. This mixture was then incubated for 10 minutes in the dark, following which the absorbance was measured at 517 nm using a spectrophotometer. Ascorbic acid was used as a positive control in this assay²¹.

Statistical analysis: Statistical analysis was carried out with the statistical package BMDP, using the BMDP 2R program (stepwise multiple regression). Results were expressed as mean of triplicate analysis.

RESULTS AND DISCUSSION

Table 1, presented the extractive values of Morinda lucida, Anogeissus leiocarpus, Sarcocephalus latifolius and their combination therapy. Extract weight in grams and yield percentage (w/w) were provided for each plant and combination therapy.

Table 1:

Extractive values of the plants singly and combination therapy

Plant	Extract weight (g)	Yield % (w/w)
Morinda lucida	9.8273	3.3
Anogeissus leiocarpus	14.7117	4.9
Sarcocephalus latifolius	14.3595	4.8
Combination therapy	13.589	4.5

Table 2:

Phytochemical analysis of the plants singly and combination therapy

	Extract
Secondary metabolites	Morinda lucida	Anogeissus leiocarpus	Sarcocephalus latifolius	Combination therapy
Carbohydrates	Positive	Positive	Positive	Positive
Tannins	Negative	Positive	Positive	Positive
Saponins	Positive	Positive	Positive	Positive
Terpenes	Positive	Positive	Positive	Positive
Sterols	Positive	Positive	Positive	Positive
Flavonoids	Positive	Positive	Positive	Positive
Alkaloids	Negative	Negative	Negative	Negative
Anthraquinones	Negative	Negative	Negative	Negative

Table 3:

Antimicrobial activity of samples at 100 mg/mL concentration against microbial isolates zone of inhibition (mm)

Extract	Streptococcus pyogenes	Staphylococcus aureus	Salmonella paratyphi	Escherichia coli	Pseudomonas aeruginosa	Klebsiella pneumoniae	Bacillus subtilis	Candida albicans
Mixed	17	17	15	10.5	13	9.5	14.5	20
AL	19	19.5	17.5	12.5	17.5	16	17	20.5
SL	9.5	-	-	-	-	-	-	9
ML	-	-	-	-	-	-	-	-
Microorganisms: Streptococcus pyogenes, Staphylococcus aureus, Salmonella paratyphi, Escherichia coli, Pseudomonas aeruginosa, Klebsiella puemoniae, Bacillis subtillis and Candida albicans. ML: Morinda lucida, SL: Sarcocephalus latifolius, AL: Anogeissus leiocarpus and MIX: Combination therapy

Anogeissus leiocarpus gave the highest extractive value of 4.9% (w/w), followed by Anogeissus leiocarpus 4.8% (w/w) and the combination therapy 4.5% (w/w) as shown in Table 1.

Table 2, outlined the results of phytochemical analysis for Morinda lucida, Anogeissus leiocarpus, Sarcocephalus latifolius and their combination therapy. It indicated the presence or absence of secondary metabolites such as carbohydrates, tannins, saponins, terpenes, sterols, flavonoids, alkaloids and anthraquinones in each extract.

Figure 1 displayed the High-Performance Liquid Chromatography (HPLC) spectrum of Morinda lucida extract showed thirteen peaks with major peaks being betulinic acid (3.526 min), chlorogenic acid (4.135 min), caffeic acid (4.577 min), rutin (6.293 min) and ferulic acid (7.495 min).

The HPLC spectrum of Anogeissus leiocarpus extract showed four peaks with major peaks being gallic acid (3.404 min), chlorogenic acid (3.859 min) and caffeic acid (4.986 min) as shown in Fig. 2.

The HPLC spectrum of Sarcocephalus latifolius extract showed six peaks with major peaks being betulinic acid (2.123 min), gallic acid (3.454 min), caffeic acid (4.411 min) and rutin (6.260 min) as shown in Fig. 3.

The HPLC spectrum of the combination therapy showed eight peaks with major peaks being betulinic acid (2.462 min), gallic acid (3.392 min), chlorogenic acid (3.830 min), caffeic acid (4.391 min) and rutin (6.252 min) as shown in Fig. 4.

Table 3, presented the antimicrobial activity of the extracts against various microbial isolates at a concentration of 100 mg/mL. The zone of inhibition (in millimeters) for Streptococcus pyogenes,

Fig. 1: HPLC spectrum of Morinda lucida extract

Fig. 2: HPLC spectrum of Anogeissus leiocarpus extract

Staphylococcus aureus, Salmonella paratyphi, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis and Candida albicans was provided for each sample.

Table 4, displayed the Minimum Inhibitory Concentration (MIC) of the extracts against selected microbial isolates. The MIC values in micrograms per milliliter (μg/mL) were listed for Streptococcus pyogenes, Staphylococcus aureus, Salmonella paratyphi, Escherichia coli, Pseudomonas aeruginosa, Klebsiella puemoniae, Bacillus subtilis and Candida albicans.

Figure 5 illustrated the DPPH free radical scavenging activities of Morinda lucida, Sarcocephalus latifolius, Anogeissus leiocarpus and the combination therapy. Key abbreviations ML, SL, AL and MIX represent Morinda lucida, Sarcocephalus latifolius, Anogeissus leiocarpus and combination therapy, respectively.

Fig. 3: HPLC spectrum of Sarcocephalus latifolius extract

Fig. 4: HPLC spectrum of the combination therapy

Table 4:

Minimum inhibitory concentration of samples against selected microbial isolates MIC (μg/mL)

Sample	Streptococcus pyogenes	Staphylococcus aureus	Salmonella paratyphi	Escherichia coli	Pseudomonas aeruginosa	Klebsiella pneumoniae	Bacillus subtilis	Candida albicans
Combinations therapy	1560	1560	1560	3125	6250	3125	1560	195.3
Anogeissus leiocarpus	781.3	195.3	781.3	1560	3125	1560	781.3	97.7
Sarcocephalus latifolius	6250	NA	NA	6250	6250	12500	12500	6250
Morinda lucida	NA	NA	NA	NA	NA	NA	NA	NA
NA: No activity

Anogeissus leiocarpus gave the highest extractive value of 4.9% (w/w), followed by Anogeissus leiocarpus 4.8% (w/w) and the combination therapy 4.5% (w/w) as shown in Table 1.

Morinda lucida, Anogeissus leiocarpus and Sarcocephalus latifolius are well-known medicinal plants with extensive traditional use in Nigeria for treating various ailments²². A combination of their leaves, prepared as a decoction, is commonly used as an herbal remedy for lymphatic filariasis and associated complications like wounds or sores^23,24.

Fig. 5: DPPH free radical scavenging activities of plant extracts and combination therapy

The extracts from the leaves of these plants were obtained through decoction and subjected to phytochemical analysis using standard methods and high-performance liquid chromatography (HPLC). Additionally, the extracts were evaluated individually and in combination for their antioxidant activity using the DPPH assay and their antimicrobial effects against a range of pathogens including Streptococcus pyogenes, Staphylococcus aureus, Salmonella paratyphi, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis and Candida albicans²⁴.

Phytochemical analysis revealed the presence of various compounds such as saponins, terpenes, sterols, phenolics and tannins in the plant extracts²⁴. The HPLC analysis further identified specific compounds in each plant extract, with major peaks corresponding to known antioxidants and bioactive compounds. Anogeissus leiocarpus extract exhibited the highest antimicrobial and antioxidant activities, followed by the combination of the three plants²⁵. These findings support the traditional use of the combination therapy for treating lymphatic filariasis, particularly regarding its antimicrobial and wound-healing properties²⁵.

Furthermore, studies on Morinda lucida and Sarcocephalus latifolius have highlighted their antibacterial, antifungal, anti-inflammatory and antimalarial properties²⁶. The phytochemical constituents of Sarcocephalus latifolius include carbohydrates, reducing sugars, tannins, saponins, flavonoids, alkaloids, steroids, glycosides, terpenoids and phenols²⁶. Sarcocephalus latifolius has been traditionally used to treat a variety of conditions such as fevers, pains, dental issues, hypertension, dysentery, diarrhea and neurological disorders²⁷. Additionally, essential oils extracted from Morinda lucida fruits exhibited antibacterial activity against various pathogens²⁸.

In summary, the combined use of Morinda lucida, Anogeissus leiocarpus and Sarcocephalus latifolius shows promise in traditional medicine for managing lymphatic filariasis and associated complications, supported by their diverse pharmacological activities and phytochemical composition^28,29. Further research is warranted to explore their full therapeutic potential and develop effective treatments based on these traditional remedies.

CONCLUSION

The extracts derived from the three plants and their combination exhibited a presence of carbohydrates, saponins, terpenes, flavonoids and sterols. Notably, Anogeissus leiocarpus demonstrated the highest levels of antimicrobial and antioxidant activity among the tested extracts. This research offers initial preclinical findings suggesting that the combination therapy may hold promise as a treatment for lymphatic filariasis (elephantiasis), pending further clinical investigations.

SIGNIFICANCE STATEMENT

The study aimed to evaluate the phytochemical profile and synergistic antimicrobial activity of Morinda lucida (ML), Anogeissus leiocarpus (AL) and Sarcocephalus latifolius (SL) leaf extracts, commonly used in Nigerian traditional medicine for addressing health issues like lymphatic filariasis. Phytochemical analysis revealed bioactive compounds such as saponins, terpenes, sterols, phenolics and tannins. High-performance liquid chromatography identified specific chemical profiles, with ML containing betulinic acid, chlorogenic acid and others, AL featuring gallic acid, chlorogenic acid and caffeic acid and SL exhibiting betulinic acid, gallic acid and caffeic acid. AL demonstrated the highest antimicrobial and antioxidant activities, followed by the combination extract, suggesting potential therapeutic benefits in treating lymphatic filariasis.

ACKNOWLEDGEMENT

The authors wish to thank the Department of Medicinal Plant Research and Traditional Medicine, National Institute for Pharmaceutical Research and Development, Idu Industrial Area, Abuja, Nigeria, for providing technical support for this study.

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