1. Introduction
Inflammation and oxidative stress play a central role in the development and progression of many chronic diseases, including hepatitis and cancer
. The inflammatory process is often accompanied by an increase in oxidative stress, caused by an overproduction of free radicals and a decrease in natural antioxidants
[2] | R. Zbadi, H. Mohti, and F. Moussaoui, “Stress oxydatif: évaluation du pouvoir antioxydant de quelques plantes médicinales,” Medecine Therapeutique, vol. 24, no. 2, pp. 134–141, Mar. 2018, https://doi.org/10.1684/met.2018.0682 |
[2]
. Oxidative stress contributes to the progression of hepatitis by promoting liver fibrosis, cirrhosis and potentially liver cancer
[3] | L. Traore et al., “Involvement of CD40 (rs1883832) and MAP3K14 (rs2074292) Genes Polymorphisms in Hepatitis B Virus Infection in Burkina Faso, West Africa,” J Biosci Med (Irvine), vol. 11, no. 12, pp. 343–361, 2023, https://doi.org/10.4236/jbm.2023.1112026 |
[3]
. For centuries, the use of medicinal plants has been the most accessible medical system in Africa. These plants contribute significantly to the life and social balance of rural African populations, especially those in the most disadvantaged areas
[4] | R. Gore and S. Gaikwad, “Checklist of Fabaceae Lindley in Balaghat Ranges of Maharashtra, India,” Biodivers Data J, vol. 3, no. 1, Mar. 2015, https://doi.org/10.3897/BDJ.3.e4541 |
[4]
. They often offer natural compounds with anti-inflammatory and antioxidant properties. However, the effectiveness of these plants can vary and it is essential to test their ability to reduce inflammation and mitigate oxidative stress
. In Burkina Faso, the use of traditional medicinal plants continues to play a crucial role in the health and well-being of populations
[6] | K. Tata Traoré et al., “African Journal of Pharmacy and Pharmacology Anti-inflammatory and analgesic activities of extracts from Balanites aegyptiaca L. Delile (Balanitaceae) root bark: Plant used against liver diseases in Bukina Faso,” vol. 13, no. 18, pp. 322–329, 2019, https://doi.org/10.9734/ajrb/2023/v13i3261 |
[6]
. Phyllanthus amarus, Cassia nigricans, Balanites aegyptiaca and Momordica charantia are among the plants widely used in traditional medicine to treat various conditions, including inflammatory complications and oxidative stress disorders
[7] | S. boria, S. G., Jean-M. C. I. P. G. André Tibiri, “Gestion des maladies hépatiques et plantes utilisées par les tradipraticiens au Burkina Faso André tibiri Noufou ouédraoGo Aude nikièma Issiaka ouédraoGo,” 2021. [Online]. Available: www.theplantlist.org |
[7]
. Phyllanthus amarus, known locally as Tchekeleni (Dioula); Tinguin garga (Mooré) is known for its hepatoprotective and anti-inflammatory properties
[8] | S. Verma, H. Sharma, and M. Garg, “Phyllanthus Amarus: A Review,” ~ 18 ~ Journal of Pharmacognosy and Phytochemistry, vol. 3, no. 2, pp. 18–22, 2014. |
[8]
. Cassia nigricans, or «Zan-drekouka» in local Mooré language is used for its antioxidant, potential anti-inflammatory and analgesic effects
[9] | O. Benjamin, J. Claude, W. Ouédraogo, E. H. Alhassan, and Y. L. Bonzi-Coulibaly, “Antioxidant activities and in vitro antimicrobial properties of cleome gynandra l. and cassia nigricans vahl extracts for water disinfection,” Annale de l’Université Joseph KI-ZERBO, 2020. https://www.researchgate.net/publication/354282058 |
[9]
. Balanites aegyptiaca, called "Kyɛglga" in the Mooré language, is known for its antimicrobial, anti-inflammatory and analgesic properties
[10] | N., J. du M. Jean-Pierre, Manuel- Plantes- Medicinales- soin- famille- Burkina-Faso, 2018th ed. 2018. |
[11] | H. N. Murthy, G. G. Yadav, Y. H. Dewir, and A. Ibrahim, “Phytochemicals and biological activity of desert date (Balanites aegyptiaca (L.) delile),” Jan. 01, 2021, MDPI AG. https://doi.org/10.3390/plants10010032 |
[10, 11]
, while Momordica charantia is renowned for its hypoglycemic, anti-inflammatory and antitumor effects
[12] | S. Jia, M. Shen, F. Zhang, and J. Xie, “Recent advances in momordica charantia: Functional components and biological activities,” Dec. 01, 2017, MDPI AG. https://doi.org/10.3390/ijms1812255 |
[12]
. The evaluation of anti-inflammatory and antioxidant recipe activity based on the association of these medicinal plants is of increasing importance in the field of pharmaceutical research and traditional medicine. Indeed, the study and identification of bioactive compounds present in these plants and their therapeutic potential could provide a solid scientific basis for their use in traditional medicine and possibly for the development of new therapeutic agents.
Thus, this study aims to demonstrate the anti-inflammatory and antioxidant activity of a recipe composed of Phyllanthus amarus, Cassia nigricans and Balanites aegyptiaca, traditionally used in the treatment of viral hepatitis B, and often associated with Momordica charantia in the treatment of liver cancer in Burkina Faso.
2. Materials and Methods
2.1. Extraction
The plant material is a recipe, Hepatib tiben, composed of dried whole plants of Phyllantus amarus and Cassia nigricans and leaves of Balanites aegyptia, prepared and packaged in capsules often associated with whole plant of dried Momordica charantia. Harvesting, drying, powder reduction and packaging are carried out by the traditional health practitioner. Infusion and maceration were the processes used for the extraction of active ingredients.
2.1.1. Infusion
A test sample of 50 g of the vegetable drug of the recipe Hepatib tiben and the powder of Momordica charantia, was placed each in a glass jar with a lid. A volume of 500 mL of boiled distilled water was poured over the contents of each jar. The mixture of the plant material and the extractor solvent was homogenized by mechanical agitation using a spatula. The homogenized mixture was kept in contact for 1 hour. The infused extract was filtered by manual pressing on a fine mesh nylon fabric. The vegetable residue after pressing was dispersed in 250 mL of distilled water and then pressed again. The resulting filtrates were filtered a second time on the nylon fabric and then centrifuged at 2000 rpm for 10 min. the supernatant of each extract was collected and the volumes were measured to determine the extraction yields. Each extract was frozen for freeze-drying.
2.1.2. Maceration
1) The hydro-alcoholic maceration
Maceration is an extraction technique that consists of mixing a dry or fresh vegetable material with an extractor solvent and macerating for a given time. The procedure used for the maceration of the Hepatib-tiben recipe was the common one. Indeed, a test sample of 50 g of the vegetable drug of the recipe Hepatib tiben was placed in a glass jar with a lid. A volume of 500ml of a water and ethanol mixture in the proportions 30:70 (v/v) was poured over the contents of the jar. The mixture of the plant material and the extractor solvent was homogenized by mechanical agitation using a spatula. The homogenized mixture was held in contact for 48 hours. The macerated extract was filtered on Watman no 5 paper. The vegetable residue was leached by percolation with small portions of the extractor solvent until clear. The resulting filtrates were concentrated by evaporation under reduced pressure to the rotary evaporator to eliminate the ethanolic phase. The residual aqueous portion of the extract after concentration was frozen for freeze-drying.
2) Successive exhaustion extraction
Successive exhaustion extraction is an extraction technique that consists of extracting until exhaustion, a dry or fresh plant material with solvents of increasing polarity. The procedure used for the fractional extraction of the Hepatib-tiben recipe was the one adopted in the laboratory. Indeed, a test sample of 100g of the vegetable drug of the recipe Hepatib-tiben was placed in a glass jar with a lid. A volume of 500ml of analytical dichloromethane (DCM) was poured over the contents of each jar. The mixture of the plant material and the extractor solvent was homogenized by mechanical agitation using a spatula. The homogenized mixture was maintained for 5 hours. The maceration was filtered on watman no 5 paper and then the vegetable residue was depleted with small portions of dichloromethane. The resulting filtrates were evaporated dry under reduced pressure using a BÜCHII rotary evaporator. The mass of the DCM dry extract obtained was determined. The vegetal residue after exhaustion with DCM was dried at laboratory room temperature and then infused with 500ml of distilled water scalded for 1 hour. The infused extract was filtered by manual pressing on a fine mesh nylon fabric. The vegetable residue after pressing was dispersed in 250ml of distilled water and then pressed again. The resulting filtrates were filtered a second time on the nylon fabric and then centrifuged at 2000 rpm for 10 min. the supernatant of the extract was collected and its volume was measured to determine the extraction yield (Rd). The infused was frozen for freeze-drying.
M: initial mass of the recipe
M': mass of dry extract after extraction and freeze-drying
3) Determination of Residual Moisture Content (THR)
The empty mass of the watch glasses was weighed and a gram of powder was added to it. The watch glasses were placed in an oven for two hours and 30 minutes. After cooling, they were weighed a second time. The Residual Moisture Content was calculated using the following formula:
P: Initial weight (g) of plant material
P’: Final weight (g) of plant material after parboiling
2.2. Phytochemical Determination of Extracts
2.2.1. Determination of Total Phenolic Content
The total phenolic content of Hepatib tiben and Momordica charantia extracts was determined using the method described by Singleton et al.
[13] | V. L. Singleton, R. Orthofer, and R. M. Lamuela-Ravent6s, “Analysis of Total Phenols and Other Oxidation Substrates and Antioxidants by Means of Folin-Ciocalteu Reagent,” 1999. |
[13]
based on the oxidizability of phenolic compounds. The reagent used was a mixture of phosphomolybdate and sodium tungstate which is reduced during the oxidation of phenols in an alkaline medium in a mixture of tungsten blue and molybdenum. There is then a change in the colorimetric properties of the Folin-Ciocalteu reagent, which reacts with the OH function of phenol. For this study, 96-well microplates were used. In each well 50µl of extract solution (1 mg/ml) were added 50µl of FCR solution (0.2N) and 100µl of sodium carbonate solution (Na
2CO
3) 20% successively. The mixture was incubated for 40 minutes in the dark, then the reading was carried out at an absorbance of 760 nm on the BioTek EPOCH 2 spectrophotometer. The results were expressed in mg Equivalent Gallic Acid per gram of dry extract (mg GAE/g ES), and calculated from the gallic acid calibration curve.
2.2.2. Determination of Total Flavonoid Content
The total flavonoid content of Hepatib tiben and Momor-dica charantia extracts was determined using the Kumaran method
[14] | A. Kumaran and R. Joel Karunakaran, “In vitro antioxidant activities of methanol extracts of five Phyllanthus species from India,” LWT, vol. 40, no. 2, pp. 344–352, 2007, https://doi.org/10.1016/j.lwt.2005.09.011 |
[14]
with some modifications. Indeed, a volume of 100 µl of 2% aluminum chloride (AlCl
3) in methanol was mixed with a volume of 100 µl of extract (1 mg/ml) diluted in methanol. The same procedure was used for quercetin, with ranges of diluted concentrations of half (from 1 mg/ml) to establish the calibration curve. Absorption was measured using the BioTek EPOCH 2 spectrophotometer at 430 nm after 30 min of light-shielded incubation. The flavonoid concentration in the extracts was derived from a calibration line established with quercetin and expressed in mg quercetin equivalent per mg extract (mg QE/g)
[15] | M. Donald Wilfried Adico, B. Bayala, B. Valérie Elvira Jean Télesphore Bazié, L. Traoré, A. Théophane Yonli, and J. Simporé, “Cytotoxic Properties on Prostate and Cervical Cancer Cells in Culture of «<i>Acti-plus</i>», a Recipe Based on Medicinal Plants from Burkina Faso,” Journal of Diseases and Medicinal Plants, Dec. 2023, https://doi.org/10.11648/j.jdmp.20230904.14 |
[15]
.
2.3. Antioxidant Activities
2.3.1. Fe3+ Ferric Ion Reduction Test (FRAP)
The ferric ion reduction test (FRAP test) is based on the ferric ion (Fe
3+) reduction in ferrous ion (Fe
2+). This method evaluates the reducing power of extracts. At low pH, when a ferric complex is reduced to the ferrous form (Fe
2+), an in-tense blue color develops with maximum absorption at 593 nm. The increase in absorbance in the reaction medium was proportional to the increase in iron reduction. The test was performed according to the method described by
[16] | I. F. F. Benzie and J. J. Strain, “The Ferric Reducing Ability of Plasma (FRAP) as a Measure of ‘“Antioxidant Power”’: The FRAP Assay,” 1996. |
[16]
. At 10 µl of extract (1mg/ml) distributed in a 96-well plate with a few wells used as blank containing 10µl of buffer, 300µl of FRAP Reagent was added: (1 volume of TPTZ (2,4,6-tripyridyl-j-triazine) + 10 volumes of CH
3COONa buffer + 1 volume of FeCl
3). Rutin was used as positive control. The reaction mixture was incubated for 30 minutes away from light and the absorbance was measured with the BioTek EPOCH 2 spectrophotometer. The absorbances were read against a standard ascorbic acid curve: equation: y = 0,8337x + 0,0499 (R2=0,9976) The results were expressed in millimole ascorbic acid equivalents per milligram (mmol EAA/mg) and calculated using the formula:
c: sample concentration (calculated from curved ascorbic acid)
D: Dilution factor
M: Molar mass of ascorbic acid (176.1g/mol)
Ci: Concentration of stock solution
The extracts and control samples were weighed and dissolved to obtain a concentration of 1 mg/ml. The test was then carried out in accordance with the established protocol for the extracts and control samples.
2.3.2. DPPH (2,2-Diphenyl-1-Picrylhydrazyl) Anti-radical Activity
The free radical scavenging capacity will be determined by the DPPH method
[17] | M. Chatatikun and A. Chiabchalard, “Phytochemical screening and free radical scavenging activities of orange baby carrot and carrot (Daucus carota Linn.) root crude extracts,” Available online www.jocpr.com |
[17]
. Each extract was prepared at 1mg/ml. The extracts were initially diluted in cascades (1/2) in plates of 96 wells. Then, 20μl of extract was mixed with 200 μl of DPPH solution (4 mg/100ml in methanol, prepared away from light). White was 20μl methanol and 200μl DPPH solution. After 30 min incubation in the dark, absorbance was read at 490 nm using the BioTek EPOCH 2 spectropho-tometer. Quercetin was used as a positive control. The meas-ured absorbance was used to calculate the percentage inhibi-tion according to the following formula:
%Inhibition=[(WhiteAbsorbance–ExtractAbsorbance)/WhiteAbsorbance]x100
The results are expressed in μg of extracts/μg of DPPH. The extract concentration capable of trapping 50% of the DPPH radicals was calculated.
2.3.3. ABTS+ (2,2’-Azinobis-(3-Ethylbenzothiazoline-6-Sulfonic Acid)) Antiradical Activity
Spectrophotometric analysis of the activity of ABTS+ rad-ical scavenging extracts was determined using the method of Re et al., 1999
[18] | R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, and C. Rice-Evans, “Original Contribution antioxidant activity applying an improved abts radical cation decolorization assay,” 1999. |
[18]
. The ABTS+ solution was prepared by dissolving 10 mg of ABTS in 2.6 ml of distilled water. Then it was added 1.7212mg of potassium persulfate and the mix-ture was kept in the dark at room temperature for 12 hours. The mixture was then diluted with ethanol to obtain an ab-sorbance of 0.70 ± 0.02 at 734 nm. In 96-well plates, 50µl of extract solution was added to 100µl of ABTS+ solution, with a blank composed of 50µL of ethanol and 100µL of ABTS solution. Mixing was carried out in 96-well plates, incubated in the dark at room temperature for 15 minutes and the concentration was read at 734 nm using the BioTek EPOCH 2 spectrophotometer. Each test was performed in triplet and the experiment was repeated three times. Inhibition Percent-ages were calculated using the following formula:
%Inhibition=[(WhiteAbsorbance–ExtractAbsorbance)/WhiteAbsorbance]x100.
The inhibition percentages were then used to calculate the concentration allowing the inhibition of 50% ABTS+ (IC50). Trolox was used as a reference compound.
2.4. Anti-inflammatory Activity in Vitro
2.4.1. Inhibition of 15-lipoxygenase (15-LOX)
The inhibition of lipoxygenase by the extracts was done according to the method of Malterud and Rydland, 2000
[19] | K. E. Malterud and K. M. Rydland, “Inhibitors of 15-lipoxygenase from orange peel,” J Agric Food Chem, vol. 48, no. 11, pp. 5576–5580, 2000, https://doi.org/10.1021/jf000613v |
[19]
. In a 96-well plate, the following reaction mixtures were prepared: the enzyme control mixture consisted of 153.75µl borate buffer plus 146.25µl lipoxygenase (400 U/ml). The mixture for enzymatic activity was composed of 3.75 µl borate buffer plus 146.25µl lipoxygenase and 150µl linoleic acid (1.25 mmol). The mixture for extraction control was composed of 146.25µl lipoxygenase, 3.75µl extract and 150 µl borate buffer. The mixture for extract activity was composed of 146.25µl lipoxygenase, 3.75µl extract and 150µl linoleic acid (substrate). Indomethacin was used as positive control. Each mixture was distributed in triplet in the wells of a 96-well plate. The plate was then read to the 234 nm absorbance spectrophotometer immediately after substrate addition. The percentage of lipoxygenase inhibition by the extracts was calculated as follows:
Inhibition (%) = [(Enzyme Absorbance - Sample Absorbance) / Enzyme Absorbance] × 100
Where,
Enzyme Absorbance = Absorbance of Enzyme Activity - Absorbance of Enzyme Blank; Sample Absorbance = Absorbance of Sample Activity - Absorbance of Sample Blank
2.4.2. Inhibition of Cyclooxygenase (COX1 & 2)
COX activity inhibition tests were performed according to the manufacturer’s procedure (Catalogue No. 560131, Cayman Chemical, Ann Arbor, MI, USA). The tests were performed on a 96-well plate. The extracts were prepared at a final concentration of 100µg/ml in the wells. A reaction mixture of 10µl extract, 10µl enzyme, 10µl hemin and 150 µl diluted buffer was distributed in 96-well plate wells. The same mixture was made without the extract but with 10µl of the solvent of dilution of the extracts and distributed in other wells. The blank consisted of 160µl of diluted buffer, 10µl of hemin and 10µl of solvent for dilution of extracts. The plate was homogenized and incubated for 5 minutes. Then, 20µl of substrate (arachidonic acid) and 20µl of colorimetric substrate were added to the wells. The plate was read at 590 nm after two-minute homogenization and incubation. The reaction mixtures were analyzed in triplet. The percentage inhibition of COX 1 & 2 was calculated using the following formula:
Inhibition (%) = [(Enzyme Absorbance - Sample Absorbance) / Enzyme Absorbance] x 100
Where,
Enzyme Absorbance = Absorbance of Enzyme Activity - Absorbance of Enzyme Blank;
Sample absorbance = absorbance of sample activity - absorbance of sample blank.
2.5. Data Analysis
All data were presented as mean standard deviation from three independent experiments. The data was entered in Excel version 2016. Statistical analyses were performed on IBM SPSS Statistics 25 for frequency calculation and one way ANOVA testing from the Prism Graph Pad 8.02 software. The Dunett test was employed to calculate p-values and perform multiple comparisons. A p-value below 0.05 was used as a criterion of statistical significance.
3. Results
3.1. Phytochemical Determination of Extracts
After extraction of the active ingredients
Hepatib tiben and
Momordica charantia, their yield was determined in relation to 100 g of dry plant matter, expressed as percentage Rd (%) (
Figure 1). The
Hepatib tiben aqueous infused yielded the highest yield 31.13 ± 0.52%, followed by its Infused-Aqueous Fraction 26.27 ± 0.19% and Hydro-Alcoholic Macerated 21.44 ± 1.96%. The
Momordica charantia aqueous infused yielded 16.95 ± 0.42%. The lowest yield was observed with the DCM Fraction of
Hepatib tiben (5.38 ± 0.03%). The residual moisture content (RH) was 4.50% and 4.99% respectively for the plant drugs
Hepatib tiben and
Momordica charantia.
Figure 1. Hepatib tiben and Momordica charantia extration yield.
I.A H. tiben: Infused-aqueous of Hepatib tiben; M. HA H. tiben: Hydroalcoholic Macerated Hepatib tiben; F. DCM H. tiben: Dichloromethane fraction of Hepatib tiben; F.I.A DCM H. tiben: Dichloromethane-infused-aqueous fraction of Hepatib tiben.
3.2. Total Phenolic Compounds Content
Table 1. Total phenolic and flavonoid content of extracts.
Drugs | Extracts | TOTAL POLYPHENOL (mg EAG/g Es) | FLAVONOIDS (mg EQ/g Es) |
Hepatib tiben | Aqueous infused | 632.53 ± 28.38a | 2.18 ± 1.69d |
Hydro-alcoholic macerate | 609.88 ± 36.57a | 2.90 ± 0.90d |
DCM Fraction | 405.83 ± 15.88d | 77.30 ± 2.03a |
Aqueous DCM Infused Fraction | 562.05 ± 18.59b | 1.23 ± 0.82d |
Momordica charantia | Aqueous infused | 513.04 ± 32.33 | 2.18 ± 0.72 |
a: non-significant difference between Hepatib tiben extracts; b: significant difference p < 0.01; d: significant difference p < 0.0001
The results of the total phenolic and flavonoid content of
Hepatib tiben and
Momordica charantia extracts are shown in
Table 1. The aqueous infused extract of
Hepatib tiben has the highest phenolic content 632.53 ± 28.38mg EAG/g Es, followed by its Hydro-alcoholic Macerate 609.88 ± 36.57 mg EAG/g Es, and its Aqueous Infused Fraction at DCM 562.05 ± 18.59 mg EAG/g Es. The DCM fraction had the lowest content of 405.83 ± 15.88 mg phenolic compounds EAG/g Es. There is a significant difference between its DCM fraction and the aqueous infused
p<0.0001. On the other hand, the flavonoid content of the DCM fraction was highest 77.30 ± 2.03 mg EQ/g Es followed by Hydro-alcoholic Macerated 2.90 ± 0.90 mg EQ/g Es and aqueous infused 2.18 ± 1.69 mg EQ/g Es. The aqueous DCM-infused extract had the lowest content 1.23 ± 0.82mg EQ/g Es with a very significant difference
p<0.0001 between the DCM fraction and the rest of the
Hepatib tiben extracts.
Momordica charantia contained 513.04 ± 32.33mg EAG/g Es and 2.18 ± 0.72mg EQ/g Es.
3.3. Antioxidant Activity
The antioxidant activity of
Hepatib tiben and
Momordica charantia was measured by three methods including FRAP, DPPH and ABTS. The results are reported in
Table 2. The ability to reduce ferric ions into ferrous ions of
Hepatib tiben extracts was evaluated at 0.01 ± 0.004mmol EAA/mg and 0,62 ± 0.01mmol EAA/mg respectively for
Hepatib tiben F. DCM and
Momordica charantia extract IA compared to the reference compound (Rutin: 2.73 ± 0.09mmol EAA/mg) with which there is a significant difference
p<0.0001 and
p=0.02 for M. HA (3.44 ± 0.01mmol EAA/mg). However, there is no difference between IA and F. IA DCM (3.58 ± 0.04mmol EAA/mg and 3.64 ± 0.02 mmol EAA/mg). The concentration of different
Hepatib tiben and
Momordica charantia extracts may inhibit 50% of DPPH
+ and ABTS
+ radicals, was evaluated and listed in
Table 2. All
Hepatib tiben and
Momordica charantia extracts had superior DPPH radical inhibitory power, with the best I. A
Hepatib tiben extract (0.002 ± 0.00001µg/ml). There is a significant difference compared to the reference compound Quercetin (3.14 ± 0.02µg/mL), p<0.0001. Regarding the inhibition of ABTS
+ cations, only F. DCM
Hepatib tiben (220.49 ± 14.53µg/ml) had a low inhibition. There is a significant difference,
p<0.0001, between this extract and the reference compound Trolox (6.17 ± 0.20µg/ml) used. The other
Hepatib tiben extracts and
Momordica charantia extract showed a rather interesting activity compared to the activity of the reference compound Trolox (6.17 ± 0.20µg/ml).
Table 2. Antioxidant activity of «Hepatib tiben» and Momordica charantia extracts.
Drugs | Extracts | FRAP (mmol EAA/mg) | DPPH IC50 (µg/ml) | ABTS IC50 (µg/ml) |
Hepatib tiben | Aqueous-infused | 3.58 ±0.04 | 0.002±0.00001d | 3.08 ±0.13a |
Hydro-alcoholic macerate | 3.44 ±0.01a | 0.004±0.0002d | 1.28 ±0.08a |
DCM Fraction | 0.01±0.004d | 0.80 ±0.066 d | 220.49±14.53d |
Aqueous DCM Infused Fraction | 3.64 ±0.02 | 0.003 ±0.0001 d | 5.19 ±0.19a |
M. charantia | Aqueous-infused | 0.62 ±0.01d | 0.012 ±0.0004 d | 15.77 ±0.66a |
| Quercetin | nd | 3.14 ±0.02 | nd |
Trolox | Nd | nd | 6.17 ±0.20 |
Rutin | 2.73 ± 0.09 | nd | nd |
M. charantia: Momordica charantia; Antioxidant activity by the ferric ion reduction test Fe3+ and by inhibition of DPPH and ABTS+ cation radicals with a: non-significant difference; d: significant difference, p < 0.0001; nd: not determined. One-Way ANOVA (and nonparametric or mixed): Dunnett’s test
3.4. Anti-inflammatory Activity in vitro
The results of the inhibitory activity of 15-LOX and COX1 & 2 by the extracts of
Hepatib tiben and
Momordica charantia are recorded in
Table 3. Comparing the values obtained with that of the reference compound Indometacin used,
Hepatib tiben hydroalcoholic maceration is the one that most inhibits 15-LOX, with an inhibition percentage of 97,72 ± 1.46%. The aqueous infused and the hydroalcoholic macerated of the same recipe has an inhibitory activity on COX 1 & 2. The values are presented in average SD (n=3). As for
Momordica charantia, it weakly inhibits 15-LOX.
Table 3. Inhibition of 15-LOX and COX 1 & 2.
Drugs | Extracts | % inhibition of 15-LOX | % inhibition of |
COX 1 | COX 2 |
Hepatib tiben | Aqueous-infused | 45.91 ± 1.3 d | 38.31 ± 0.44d | 30.19 ± 0.07d |
hydroalcoholic macerated | 97.72 ± 1.46 b | 24.05 ± 0.014d | 36.29 ± 3.74d |
M.charantia | Aqueous-infused | 16.66 ± 0.93 d | nd | nd |
| Indomethacin | 92.25 ± 0.90 | nd | nd |
M. charantia: Momordica charantia; 100µg/mL of extract was used for each test; %: percentage; c: significant difference p < 0.001; d: significant difference p < 0.0001; nd: not determined; Indomethacin: 100µg/mL. One-Way ANOVA (and nonparametric or mixed): Dunnett’s test
4. Discussion
African flora displays a very huge and an important bio-diversity which has been used as main source of medicines for millennia. In fact, more than 80% of the African population uses plants to treat from various ailments to important dis-eases due the accessibility of plants and the cultural values attached to them
[20] | W. R. Sawadogo, M. Schumacher, M. H. Teiten, M. Dicato, and M. Diederich, “Traditional West African pharmacopeia, plants and derived compounds for cancer therapy,” Nov. 15, 2012, https://doi.org/10.1016/j.bcp.2012.07.021 |
[20]
. Traditional health practitioners also work in collaboration with modern medicine actors, ministries, to strengthen their capacity and to validate information and practices on the judicious use of herbal remedies
[21] | Organisation Ouest-Africaine de la Santé (OOAS), “PHARMACOPÉE D’AFRIQUE DE L’OUEST,” 2013. |
[21]
. Recipes are developed by traditional health practitioners to treat several pathologies including viral hepatitis B and cancer. It is also a recipe of Hepatib tiben plants used by a traditional health practitioner in Burkina Faso that was the subject of this study. This recipe composed of whole plants of Phyllanthus amarus and Cassia nigricans then leaves of Balanites aegyptiaca is used in the management of viral hepatitis B, and is associated in some cases with Momordica charantia to treat cases of liver cancer by this traditional health practitioner from Burkina Faso.
The aqueous infusion of Hepatib tiben had the highest ex-traction yield (31.13 ± 0.52%) reflecting the high solubility of the active ingredients of Hepatib tiben in water than in other extraction solvents used. The residual moisture content (THR) of both plant drugs is less than 10%. This means that the plant material of both drugs was dried under acceptable conditions according to the standards of the European Pharmacopoeia (2010 edition), with a low risk of mold formation, signs of oxidation and deterioration of the active ingredients
[15] | M. Donald Wilfried Adico, B. Bayala, B. Valérie Elvira Jean Télesphore Bazié, L. Traoré, A. Théophane Yonli, and J. Simporé, “Cytotoxic Properties on Prostate and Cervical Cancer Cells in Culture of «<i>Acti-plus</i>», a Recipe Based on Medicinal Plants from Burkina Faso,” Journal of Diseases and Medicinal Plants, Dec. 2023, https://doi.org/10.11648/j.jdmp.20230904.14 |
[15]
.
Plant phenolic compounds are one of the main groups of compounds that act as antioxidants and anti-inflammatories
[22] | M. Sawadogo, M. J. Bangou, A. Lema, M. H. Thiombiano, H. Y. Ouoba, and B. Ouoba, “Medicinal Plants Used in Composition of Anti-Viral Hepatitis Recipes in Burkina Faso: Ethnobotanical Study,” European Journal of Pharmaceutical Research, vol. 2, no. 2, pp. 1–10, Jul. 2022, https://doi.org/10.24018/ejpharma.2022.2.2.34 |
[22]
. Phytochemical analysis of Hepatib tiben and Momor-dica charantia extracts revealed the presence of phenolic compounds in all extracts. The aqueous infused extract of Hepatib tiben had the highest content of total phenolic com-pounds (632.53 ± 28.38 mg EAG/g Es) and DCM Fraction in flavonoids (77.30 ± 2.03 mg EQ/g Es). The results obtained are similar to those of several authors
[8] | S. Verma, H. Sharma, and M. Garg, “Phyllanthus Amarus: A Review,” ~ 18 ~ Journal of Pharmacognosy and Phytochemistry, vol. 3, no. 2, pp. 18–22, 2014. |
[11] | H. N. Murthy, G. G. Yadav, Y. H. Dewir, and A. Ibrahim, “Phytochemicals and biological activity of desert date (Balanites aegyptiaca (L.) delile),” Jan. 01, 2021, MDPI AG. https://doi.org/10.3390/plants10010032 |
[8, 11]
who also showed the presence of the same compounds in the extracts of Phyl-lanthus amarus and Balanites aegyptiaca (L.) Delile, plants that make up the recipe Hepatib tiben. The work of
[23] | J. Yakubu, O. A. Sodipo, and S. Umarfaruk Abubakar, “Phytochemical Profiling, Toxicity Study and Abortifacient Activity of Seed and Whole Plant of Momordica charantia Linn. (Cucurbitaceae),” 2022. |
[23]
isolated flavonoids in the methanolic extract of Momordica charantia. The variation in the amount of these secondary metabolites observed between the different extracts of Hepatib tiben could be explained by the nature of the extraction solvents. Polyphenolic compounds are more soluble in high-polarity solvents such as water, while flavonoids are more soluble in low-polarity solvents such as dichloromethane
[24] | A. R. Abubakar and M. Haque, “Preparation of medicinal plants: Basic extraction and fractionation procedures for experimental purposes,” Jan. 01, 2020, Wolters Kluwer Medknow Publications. https://doi.org/10.4103/jpbs.JPBS_175_19 |
[25] | M. D. W. Adico, B. Bayala, J. Bunay, S. Baron, J. Simpore, and J. M. A. Lobaccaro, “Contribution of Sub-Saharan African medicinal plants to cancer research: Scientific basis 2013–2023,” Apr. 01, 2024, Academic Press. https://doi.org/10.1016/j.phrs.2024.107138 |
[24, 25]
. In one study, water was found to be the best solvent for the extraction of phenolic compounds from Phyllanthus amarus, resulting in a higher yield and total phenolic content than other solvents such as ethanol, methanol and dichloromethane
[26] | V. T. Nguyen, H. N. T. Pham, M. C. Bowyer, I. A. Van Altena, and C. J. Scarlett, “Influence of solvents and novel extraction methods on bioactive compounds and antioxidant capacity of Phyllanthus amarus,” Chemical Papers, vol. 70, no. 5, pp. 556–566, May 2016, https://doi.org/10.1515/chempap-2015-0240 |
[26]
. The high flavonoid content for the DCM fraction could be explained by the presence of methoxylated and hydroxylated aglycones, which are low polarity compounds with an affinity for dichloromethane
[27] | U. K. Merbah-Ouargla et al., “Extraction des Flavonoïdes et évaluation de l’efficacité Antioxydant d’une plante algérienne,” 2019. |
[27]
.
Oxidative stress, involved in many chronic diseases such as cardiovascular diseases, cancer, neurodegenerative diseases, refers to an imbalance between the production of free radicals and the body’s ability to neutralize their harmful effects with antioxidants. Free radicals are highly reactive molecules that can damage cells, proteins and DNA if they are not effectively neutralized. The ability of Hepatib tiben and Momordica charantia extracts to prevent oxidation was tested by three complementary methods: the reduction of ferric ion (Fe
3+) to ferrous ion (Fe
2+) (FRAP), the ability to trap DPPH radicals and ABTS+ cation radicals, in order to better appreciate the antioxidant activity of the extracts. All extracts showed a rather interesting antioxidant activity through the anti-radical activity by the three methods. For Momordica charantia, the results corroborate those of Yakubu et al., (2022) who found an IC50 = 20.63μg/ml for the radical ABTS+. The aqueous infusion Hepatib tiben showed the best antioxidant activity with the methods by DPPH, ABTS and FRAP which exceeded those of Quercetin, very close to that of Trolox and Rutin, used as reference compounds. F. DCM and Momordica charantia had the best antioxidant activities by the FRAP method. These interesting antioxidant activities of the extracts of Hepatib tiben and Momordica charantia in general could be explained by the fact that these drugs are a cocktail of plants containing phenolic compounds and fla-vonoids that are known for their antioxidant effect
[8] | S. Verma, H. Sharma, and M. Garg, “Phyllanthus Amarus: A Review,” ~ 18 ~ Journal of Pharmacognosy and Phytochemistry, vol. 3, no. 2, pp. 18–22, 2014. |
[9] | O. Benjamin, J. Claude, W. Ouédraogo, E. H. Alhassan, and Y. L. Bonzi-Coulibaly, “Antioxidant activities and in vitro antimicrobial properties of cleome gynandra l. and cassia nigricans vahl extracts for water disinfection,” Annale de l’Université Joseph KI-ZERBO, 2020. https://www.researchgate.net/publication/354282058 |
[11] | H. N. Murthy, G. G. Yadav, Y. H. Dewir, and A. Ibrahim, “Phytochemicals and biological activity of desert date (Balanites aegyptiaca (L.) delile),” Jan. 01, 2021, MDPI AG. https://doi.org/10.3390/plants10010032 |
[8, 9, 11]
. These effects are beneficial to health, including the man-agement of oxidative stress and the prevention of various related diseases.
15-lipoxygenase and cyclooxygenases 1 and 2 are enzymes involved in arachidonic acid cascade and eicosanoid biosynthesis, but catalyze different reactions and play distinct roles in regulating inflammatory response and other biological processes
[28] | S. A. Adebayo, J. P. Dzoyem, L. J. Shai, and J. N. Eloff, “The anti-inflammatory and antioxidant activity of 25 plant species used traditionally to treat pain in southern African,” BMC Complement Altern Med, vol. 15, no. 1, Dec. 2015, https://doi.org/10.1186/s12906-015-0669-5 |
[28]
. When comparing the results obtained with those of indomethacin which is the reference compound used, the Hepatib tiben Hydroalcoholic Macerate has a good ability to inhibit 15-LOX with inhibition percentages of 97.72 ± 1.46%. A study analyzed the ethanolic and aqueous extracts of Balanites aegyptiaca, one of the plants that make up Hepatib tiben, and showed moderate activity (41-70% inhibition) in the inhibition of 15-LOX
[6] | K. Tata Traoré et al., “African Journal of Pharmacy and Pharmacology Anti-inflammatory and analgesic activities of extracts from Balanites aegyptiaca L. Delile (Balanitaceae) root bark: Plant used against liver diseases in Bukina Faso,” vol. 13, no. 18, pp. 322–329, 2019, https://doi.org/10.9734/ajrb/2023/v13i3261 |
[6]
. Indeed, the hydroalcoholic maceration of Hepatib tiben has as high a content of phenolic compounds as the aqueous infused, and also has bioactive ingredients that inhibit 15-LOX
[28] | S. A. Adebayo, J. P. Dzoyem, L. J. Shai, and J. N. Eloff, “The anti-inflammatory and antioxidant activity of 25 plant species used traditionally to treat pain in southern African,” BMC Complement Altern Med, vol. 15, no. 1, Dec. 2015, https://doi.org/10.1186/s12906-015-0669-5 |
[28]
. This high content of phenolic compounds could justify here the best anti-inflammatory activity by inhibition of the 15-Lox of the hydroalcoholic extract of Hepatib tiben. Most of the plants that make up these recipes are traditionally used for their anti-inflammatory properties
[7] | S. boria, S. G., Jean-M. C. I. P. G. André Tibiri, “Gestion des maladies hépatiques et plantes utilisées par les tradipraticiens au Burkina Faso André tibiri Noufou ouédraoGo Aude nikièma Issiaka ouédraoGo,” 2021. [Online]. Available: www.theplantlist.org |
[7]
.
COX1 is naturally present in cyclooxygenases and main-tains tissue integrity, while COX2 is induced in response to inflammation
[29] | C. Sobolewski, “Effets d’inhibiteurs de la cyclooxygénase-2 sur la prolifération et la survie de cellules cancéreuses hématopoïétiques,” HAL open science, 2018, [Online]. Available: https://hal.univ-lorraine.fr/tel-01746243 |
[29]
. The aqueous and hydroalcoholic infused extracts of Hepatib tiben show an inhibition of both forms of COX. COX2 plays a crucial role in inflammation by producing prostaglandins that amplify the inflammatory response. However, simultaneous inhibition of both forms can not only reduce overall inflammation but also cause gastrointestinal side effects due to inhibition of COX1, which is associated with gastric disorders
. Indeed, the plants from the recipes Momordica charantia and Hepatib tiben are rich in phenolic and flavonoid compounds
[6] | K. Tata Traoré et al., “African Journal of Pharmacy and Pharmacology Anti-inflammatory and analgesic activities of extracts from Balanites aegyptiaca L. Delile (Balanitaceae) root bark: Plant used against liver diseases in Bukina Faso,” vol. 13, no. 18, pp. 322–329, 2019, https://doi.org/10.9734/ajrb/2023/v13i3261 |
[8] | S. Verma, H. Sharma, and M. Garg, “Phyllanthus Amarus: A Review,” ~ 18 ~ Journal of Pharmacognosy and Phytochemistry, vol. 3, no. 2, pp. 18–22, 2014. |
[31] | A Rafidah Binti A Mohd Yunos, “Inhibition of cyclooxygenase and lipoxygenase activities of Momordica charantia,” 2019. |
[32] | S. Osunga, O. Amuka, A. K. Machocho, A. Getabu, and M. O. Onani, “Some members of genus Cassia (Senna): Their Ethnobotany, Potency and Prospects of Drug discovery,” Indian J Pharm Drug Studies, vol. 2, no. 2, p. 41, Jun. 2023. |
[6, 8, 31, 32]
with anti-inflammatory properties that modulate the expression of COX and inhibit the release of pro-inflammatory mediators
. Studies have shown that methanolic extracts of Phyllanthus amarus significantly inhibit the production of pro-inflammatory mediators such as TNF-α, IL-1β and PGE2, as well as the expression of COX2 in human macrophages
[33] | S. Doumbia, A. Denou, M. Haidara, D. L. Dembele, N. B. Y. Fofie, and R. Sanogo, “A review on medicinal plants used in the management of liver diseases in West Africa,” Journal of Pharmacy & Bioresources, vol. 20, no. 2, pp. 56–73, Jun. 2023, https://doi.org/10.4314/jpb.v20i2.1 |
[33]
. These results suggest that aqueous infusions of Hepatib tiben and Momordica charantia could be promising due to their high content of total phenolic compounds, known for their anti-inflammatory properties through inhibition of enzymes 15-LOX, COX1 & COX2. Indeed, authors have obtained similar results with extracts of Balanites aegyptiaca, a plant part of the recipe Hepatib tiben
[6] | K. Tata Traoré et al., “African Journal of Pharmacy and Pharmacology Anti-inflammatory and analgesic activities of extracts from Balanites aegyptiaca L. Delile (Balanitaceae) root bark: Plant used against liver diseases in Bukina Faso,” vol. 13, no. 18, pp. 322–329, 2019, https://doi.org/10.9734/ajrb/2023/v13i3261 |
[6]
. Given the link between inflammation and cancer, these results could justify the combined use of Hepatib tiben and Momordica charantia for the treatment of advanced chronic viral hepatitis and liver cancer by traditional health practitioners in Burkina Faso
[6] | K. Tata Traoré et al., “African Journal of Pharmacy and Pharmacology Anti-inflammatory and analgesic activities of extracts from Balanites aegyptiaca L. Delile (Balanitaceae) root bark: Plant used against liver diseases in Bukina Faso,” vol. 13, no. 18, pp. 322–329, 2019, https://doi.org/10.9734/ajrb/2023/v13i3261 |
[6]
. In light of these results, it would be highly advantageous to use Hepatib tiben due to its high polyphenol and flavonoid content. These secondary plant metabolites have demonstrated their antioxidant capacity through FRAP, DPPH, and ABTS methods, as well as their anti-inflammatory capacity through the inhibition of 15 lipoxygenase.
Author Contributions
Pengdwendé Fabienne Ingrid Zongo: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Project administration, Software, Validation, Visualization, Writing – original draft, Writing – review & editing
Bagora Bayala: Conceptualization, Data curation, Formal Analysis, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing
Luc Zongo: Validation, Visualization, Writing – review & editing
Albert Yonli: Validation, Visualization
Jean Marie Kabore: Validation, Visualization
Jacques Simpore: Conceptualization, Methodology, Resources, Validation, Visualization