|Year : 2022 | Volume
| Issue : 2 | Page : 53-59
The Nigerian Chikadoma plant: Formulation and evaluation of an herbal anti-inflammatory and antimicrobial gel containing yellow bush (Duranta repens) leaf extract
Babatunde A S Lawal1, Linus K Eban1, Godwin Christian Akuodor2, Sylvester C Ohadoma1
1 Department of Pharmacology, Faculty of Basic Medical Sciences, College of Medical Sciences, University of Calabar, Calabar, Nigeria
2 Department of Pharmacology and Therapeutics, Faculty of Medicine, College of Health Sciences, Nnamdi Azikiwe University, Nnewi Campus, Awka, Nigeria
|Date of Submission||04-Jul-2022|
|Date of Decision||15-Aug-2022|
|Date of Acceptance||18-Aug-2022|
|Date of Web Publication||27-Oct-2022|
Dr. Sylvester C Ohadoma
Department of Pharmacology, Faculty of Basic Medical Sciences, College of Medical Sciences, University of Calabar, Calabar
Source of Support: None, Conflict of Interest: None
Introduction: A plant commonly referred to as Chikadoma in Nigeria, yellow bush in English, is traditionally used for the treatment of microbial infections and inflammation. Objectives: This study evaluated a novel gel formulation of the Nigerian Chikadoma plant extract for its topical antimicrobial and anti-inflammatory activities against pure clinical isolates (Bacillus subtilis, Salmonella paratyphi, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger), and carrageenan-induced edema on experimental rodents, respectively. Materials and Methods: Concentration of 1% w/w Carbopol-934 was employed as the gelling agent. The physiochemical parameters of formulations (viscosity, pH, spreadability, extrudability, and stability) were examined for a month to ensure that when applied on the skin, it never produced any skin irritation/erythema/edema. Results: The physiochemical and stability assessment of the gel formulation showed viscosity, pH, spreadability, extrudability, and stability within standard specifications. The antimicrobial studies showed activity against some of the test microorganisms, while the carrageenan-induced edema was inhibited. Both showed high degree of reproducibility. Conclusion: The formulated gel of Nigerian Chikadoma plant extract significantly possesses herbal anti-inflammatory and antimicrobial effects, and the gel preparation was stable under normal storage environment and did not manifest skin irritation/erythema and edema when applied on the skin.
Keywords: Anti-inflammation, antimicrobial, Chikadoma plant, Duranta repens, herbal gel, yellow bush
|How to cite this article:|
Lawal BA, Eban LK, Akuodor GC, Ohadoma SC. The Nigerian Chikadoma plant: Formulation and evaluation of an herbal anti-inflammatory and antimicrobial gel containing yellow bush (Duranta repens) leaf extract. Niger J Exp Clin Biosci 2022;10:53-9
|How to cite this URL:|
Lawal BA, Eban LK, Akuodor GC, Ohadoma SC. The Nigerian Chikadoma plant: Formulation and evaluation of an herbal anti-inflammatory and antimicrobial gel containing yellow bush (Duranta repens) leaf extract. Niger J Exp Clin Biosci [serial online] 2022 [cited 2022 Dec 10];10:53-9. Available from: https://www.njecbonline.org/text.asp?2022/10/2/53/359780
| Introduction|| |
Synthetic chemicals are widely used for the management of various disease conditions including microbial infections and inflammation. Because of poverty specifically, coupled with the development of allergic reaction, immunity suppression, and resistance to these commonly used drugs, low- and middle-income countries like Nigeria resort to medicinal plants as alternative agents, necessitating the formulation and evaluation of herbal extracts. Recent reports make it more threatening and frightening that late presentation to care in low- and middle-income countries may suggest that people living with deadly diseases such as HIV/AIDs may express pronounced immunosuppression and chronic inflammation even at antiretroviral therapy initiation., The use of plant extracts is less damaging to the human health and environment because the secondary metabolites occur as complexes of naturally and structurally related analogues. In Nigeria, a plant commonly referred to as Chikadoma also known as Yellow bush is employed in the folkloric management of various ailments such as microbial infections, pain, and inflammation. It belongs to the Family of Verbenaceae with botanical name Duranta repens. Other common English name is “Geisha girl.” It is planted widely in homes, schools, hospitals, and various social rendezvous as ornamental plant. It is easily recognized as a bushy shrub [Figure 1] with bright yellow, sweet-smelling flowers blended with purple and white colors, that grows up to 6 feet (1.8 m) tall and native to Japan. Many plant species that are being used nowadays have been utilized for centuries in a localized part of the world but the rise in global migration and communications have favored many of these currently being used worldwide. Phytomedicine is currently available in most countries as food supplement. No doubt, the practice of herbal medicine has achieved continuous patronage making it an inevitable global discourse. Finished herbal products are packaged in different dosage forms such as herbal powders, teas, alcoholic beverages, decoctions, tablets, capsules, creams, ointments, and gels. Huge scale production due to the growing demand for herbal medicinal products may be expected which accordingly may lead to longer storage times and envisaged product deterioration. As a precaution, stability studies become necessary in the determination of shelf-life and fortification of the quality of plant product. Physical or sensory tests, chemical or chromatographic/spectral test, and microbiological tests may be involved in stability studies. Yes, chemical stability determination of a herbal preparation is usually challenging recognizing that a plant extract may retain many different compounds. In addition to that fact, plant enzymes including glycosidases, oxidases, or esterases play important role in the decomposition of secondary metabolites of plant. This study dwelled on gel dosage form of herbal leaf extract of Chikadoma plant (yellow bush) and its stability considerations.
| Materials and Methods|| |
Plant collection and preparation of leaf extract
Fresh stalks of Chikadoma plant, Yellow bush (D. repens) were collected from environs of the University of Calabar, Nigeria, in July 2021 and authenticated by Dr. Ebigwai Joseph of the Department of Botany, University of Calabar, where a voucher number: Bot/Herb/Ucc/065 has been deposited in the Herbarium. The fresh leaves of the plant were plucked from the stem and air-dried at room temperature (26°C). The dried leaves were pulverized into powder using an electric blender. The powdered leaves (200 g) were extracted with methanol by cold maceration for 48 h and filtered to obtain the methanol extract. With the use of rotatory evaporator at reduced pressure, the extract was concentrated and further oven-dried. The extract was subjected to phytochemical analysis using standard methods.,
Young healthy male albino rabbits, weighing 1.5–2 kg, were used at the start of the experiment. Later, albino Wistar rats (150–200 g) were employed for evaluation of anti-inflammatory activity. The experimental animals were housed together in a clean tank spacious enough to facilitate free movement and accommodation to hold feed and drinking water.
About 24 h before the anti-inflammatory test, the fur were removed by closely clipping the dorsal area of the trunk of the animals, taking care to avoid abrading the skin. Only healthy, intact skin animals were employed in the study.
Formulation of topical gel preparation
The herbal gel was prepared using Carbopol-934 as the gelling agent in 1% w/w concentration with water (deionized) using mechanical stirrer. It was necessary to adjust the pH of the gel to neutral by adding small amount of triethanolamine with continuous stirring. Herbal extract (1% w/w) of yellow bush was added to the gel and stirred for homogenous mixing of the extract in gel base, before filling into collapsible tubes and subsequently stored in a cool, dry place. Physical parameters of appearance, color, and feeling on application were recorded.
The determination of stability relied on ICH guidelines. The gel was filled in collapsible tubes and kept at different humidity and temperature conditions such as 25 ± 2 °C/60 ± 5% RH, 30 ± 2 °C/65 ± 5% RH, 40 ± 2 °C/75 ± 5% RH spanning a period of 3 months and studied for pH, appearance, and spreadability.
The pH of the gel was measured using a pH meter and recorded.
The viscosity of the gel preparation was measured using Brookfield viscometer with spindle #7, and recorded.
The gel formulation was filled in capped collapsible tubes and the weight recorded. The extrudability record was achieved by placing the collapsible filled tube between two glass slides and then the cap would be removed. The quantity of the extruded gel was collected and weighed. The percentage of the extruded gel was calculated. >90%, >80%, and >70% of extrudability are considered excellent, good, and fair, respectively.,
This was determined using the apparatus that consist of a wooden block, which was provided by a pulley at one end. This method measures spreadability on the basis of slip and drag characteristics of gel. About 2 g excess of gel under study was placed on the ground slide, and the gel sandwiched between the slide and another glass slide having a dimension of fixed ground slide and provided with a hook. A weight (1 kg) was placed on the top of the two slides for 5 min to expel air and to avail a uniform film of the gel between the slides. The gel in excess was scrapped off from the edges. The time in seconds required by the two slides to cover a distance of 7.5 cm when the top plate was subjected to pull of 80 g with the help of string attached to the hook, was noted and recorded. A better spreadability was indicated when a short interval was noted.,
The following formula was used to calculate spreadbaility: S = M × L/T
where S = spreadability; M = weight in the pan (tied to the upper slide); L = length moved by the glass slide; T = time (in seconds) taken to separate the upper slide from the ground slide.
Primary dermal irritation index
The manifestation of reversible damage to the skin as a result of the application of a test substance for up to 4 h is referred to as dermal irritation. Primary dermal irritation index (PDII) is a methodology for classifying topical formulations into different categories based on acute toxic reactions observed upon single application of a formulation on skin.
Such formulation can be graded as irritating or nonirritating depending on the PDII score (<0.5: nonirritating, 0.5–2.0: slightly irritating; 2.1–5.0: moderately irritating, and 5.0: severely irritating).
Herbal gel application
As the test substance, half a gram of the Yellow bush herbal gel was applied to an area of approximately 6 cm2 of the animal skin and covered with a gauze patch, which was loosely held in contact with the skin using a suitable semi-occlusive dressing for 4 h and then removed. After 4 h of exposure period, residual test substance was removed without changing the existing response or integrity of the epidermis. After 1 h of the removal of the patch, observations were recorded. The same was repeated for control animals (that is, 0.5 g of the gel base without the herbal mixture).
Simultaneously, both the test and control animals were observed daily for manifestation of skin irritation/toxic reactions (edema or erythema). A value between 0 and 4, was assigned for every observation of the skin, where 0 means no eschar formation and skin erythema, and 1, 2, 3, and 4 stand for every slight, well defined, moderate, and severe erythema to eschar formation, respectively. The same would stand for 0: no edema and 4: severe edema.
Repeated dose dermal toxicity of the developed herbal gel formulation
Twenty eight days repeated dose dermal toxicity of the yellow bush herbal gel formulation was conducted on the rabbit skin to ascertain the cumulative toxicity tendencies upon repeated application, to include behavioral, and biochemical parameters. These were done alongside the control study, for any manifestation of erythema or edema.
Blood samples were collected separately from each control and experimental animal by retro orbital puncture on the 14th and 28th day of the study, for the determination of such parameters as blood sugar, creatinine, urea, total and direct bilirutin, total cholesterol, protein, alkaline phosphatase, and acid phosphate, etc.
Albino Wistar rats of either sexes (150–200 g) were used. Three groups of six animals each were used following all animal care procedures, in the induction of edema using carrageenan. Before the experimentation, the animals were fasted for 24 h with water ad libitum. From 1% suspension of carrageenan in normal saline prepared 1 h before each experiment, approximately 50 μL was injected into the plantar side of right hind paw of rats. Herbal gel (0.2 g) containing 1% yellow bush extract was applied by gentle rubbing 50 times with the index finger on the plantar surface of the hind paw of animals in Group II.
Group I received plain gel base serving as control (negative), and
Group III received 0.2 g l % valdecoxib gel serving as the standard (positive control).
All medicaments were applied topically 1 h before the carrageenan injection. After which the paw volume was measured immediately after carrageenan injection using a plethysometer, at 1, 2, 3, and 4 h intervals.
Pure clinical isolates of Pseudomonas aeruginosa cillus subtilis, Salmonella More Details paratyphi, Escherichia More Details coli, Staphylococcus aureus, Candida albicans, and Aspergillus niger were obtained from medical laboratory unit of University of Calabar Teaching Hospital, Calabar, Nigeria.
Determination of sensitivity and inhibitory zone diameter (IZD)
The modified agar-well diffusion technique was employed. Each of the test organisms was streaked on the surface of the different sterile sensitivity agar media. Wells were bored on the agar media using sterile cork borer of 6 nm diameter. Exactly 2 drops of the extract prepared as described earlier were accordingly put into the wells and then allowed to stand for 30 min for proper diffusion. Standard drugs (ampicillin 80, tetracycline 40, gentamicin, and ciprofloxacin 40 μg/ml) were served as control. The plates were then incubated aerobically at 37°C for 2 h.
Determination of minimum inhibitory concentration
Minimum inhibitory concentration (MIC) was determined using the microbroth dilution technique. The extract and fractions were incorporated at varying concentrations into nutrient broth, respectively, containing the growth medium and each of the test organisms, excluding the incubated at 37°C for 24 h. The lowest concentration of extract and fraction that did not allow microbial growth within the incubation period was taken to be the MIC.
Data were expressed as the mean ± standard error of mean and performed by means of analysis of variance, followed by Student's t-test. P < 0.05 was considered statistically significant.
| Results|| |
The phytochemical studies revealed that methanol leaf extract of Nigerian Chikadoma plant had the abundance of alkaloids, glycosides, steroids, saponins, terpenes, and flavonoids. Resins and protein occurred in moderate amounts, while reducing sugar appeared but in trace amount [Table 1].
Nigerian Chikadoma leaf extract has activity against the test bacteria except E. coli and no significant activity against fungi. The MIC of the extract on the four microorganisms that showed sensitivity and inhibitory zone diameter (IZD) are as shown in the [Table 2], while the results of agar diffusion bioassay of the diluted standard drugs (ciprofloxacin, ampicillin, tetracycline, and gentamicin) for MIC determination against the microorganisms that showed susceptibility are as shown in the [Table 3], [Table 4], [Table 5], [Table 6].
|Table 2: Sensitivity test and inhibitory zone diameter2 (mm2) of crude methanol leaf extract of Nigeria Chikadoma plant|
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|Table 3: Result of inhibitory zone diameter (mm) and inhibitory zone diameter2 (mm2) of ciprofloxacin (concentration of stock=40 g/ml)|
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|Table 4: Result of inhibitory zone diameter (mm) and inhibitory zone diameter2 (mm2) of ampicillin (concentration of stock=80 μg/ml)|
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|Table 5: Result of inhibitory zone diameter (mm) and inhibitory zone diameter2 (mm) of tetracycline (concentration of stock=40 g/ml)|
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|Table 6: Result of inhibitory zone diameter (mm) and inhibitory zone diameter2 (mm2) of gentamicin (concentration of stock=40 g/ml)|
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The anti-inflammatory tendency produced after topical administration of Chikadoma herbal gel formulation on carrageenan-induced hind paw edema showed a high degree of reproducibility [Table 7].
|Table 7: Effect of topical application of Chikadoma herbal gel on carrageenan-induced rate paw edema|
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Formulation of Chikadoma gel preparation
The physical parameters of appearance, color and feeling on application of Chikadoma herbal gel presented greenish in color with a translucent appearance which has a cooling sensation during the period of evaluation. The pH was 6.8 and did not produce any sign of irritation on the skin application.
Extrudability and spreadability
Extrudability was excellent [Table 8] and spreadability showed less variant [Table 9] after undertaking stability studies from that of the initially prepared gel.
|Table 8: Extrudability of Chikadoma herbal gel at the time of preparation (mean±standard error of mean)|
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|Table 9: Spreadability of Chikadoma herbal gel during the evaluation period (mean±standard error of mean)|
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Viscosity and stability
The initial viscosities were noted at 25°C [Table 10]. Subsequently, the stability study's results showed that the preparation was stable under normal storage environment.
Primary dermal irritation index
The PDII of the formulation was 0.00 and can be classified as nonirritant to the rabbit skin.
| Discussion|| |
The result of this study revealed that the gel formulation of Nigerian Chikadoma leaves extract was stable under normal storage conditions and never manifested any skin irritation/erythema or edema. This is in consonance with the ICH guidelines; spanning a period of 3 months and studied for pH, appearance, spreadability, PDII, extrudability. The formulation can be safely graded as “nonirritating.” PDII support this observation, as a method of classifying topical formulations into various categories based on acute toxic reactions notable upon single application on the skin. Dermal irritation manifests reversible damage to the skin following the application of a test substance for up to 4 h. Chikadoma plant showed appreciable activity against S. aureus, P. aeruginosa, B. subtilis, and S. paratyphi, but manifested weak activity on A. niger and C. albicans; hence, it may be a broad-spectrum antimicrobial. Extrapolations regarding the graph of IZD2 (mm)2 against log concentration of extract and standard antimicrobials gave their MIC values. Observation indicated that the greater the IZD produced, the lower the MIC hence the more potent the agent. The extract and standard antimicrobials had varying MICs on individual microorganisms. When the activity of the extract on B. subtilis was compared with those of standard drugs, it was noticed that gentamicin with MIC of 0.02 μg/ml showed the highest activity. This was followed by ciprofloxacin (0.02 μg/ml) tetracycline and finally ampicillin with MIC 1.33 and 5.62 μg/ml, respectively, upon the high stock concentration. The Gram-positive bacteria showed more susceptibility to Chikadoma extract and standard drugs than the Gram-negative bacteria even the resistance by E. coli. These findings is in agreement with the susceptibility of the microorganisms to different plant extracts as reported by researchers which could be explained by the tendency of cell wall of Gram-positive bacteria being less complex and lack the natural sieve effect against large molecules.,, The extract showed no activity on E. coli as well as weak activity against the test fungi, that is C. albican and A. niger. The high content of phytochemicals could be the basis of its antimicrobial action which is in agreement with the reports that plants rich in saponins and tanins have antimicrobial properties. The anti-inflammatory observation in this study could be attributed to the plethora of phytochemicals also, such as flavonoids which are very useful antioxidant suggesting the plant importance in the treatment of inflammation and tumor.
| Conclusion|| |
The results and data presented in this study demonstrate that gel form of leaf extract of a plant commonly referred to as “Chikadoma plant” in Nigeria, “Yellow bush” in English, possess significant topical antimicrobial and anti-inflammatory properties, giving credence to their traditional use for the treatment. Further studies are encouraged in order to isolate and characterize the various active principle (s) responsible for the bioactivities.
- SCO conceived and designed the study and BASL carried out the literature search,
- BASL, LKE, and GCA carried out the experiments.
- SCO and BASL wrote the draft manuscript.
- All of the authors read and approved the final manuscript for publication.
The study protocol was carried out in accordance with the rules and regulations of the Institutional Animal Ethical Committee, Faculty of Basic Medical Sciences, University of Calabar, Calabar, Nigeria (UC/FBMS/21/033), as well as the international guidelines on the Use and Handling of Experimental Animals.
Financial support and sponsorship
This study was funded by the authors.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10]