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In vitro Anti-inflammatory and in vivo Immuno-modulatory Activities of Ethanolic Extract of Strychnos camptoneura (Loganiaceae) Seeds

Morabandza Cyr Jonas*, Moulari Brice, Gombé Assoungou Hermann, Abena Ange Antoine

Laboratory of Microbiology, infectiology and Immunology, higher normal school, Marien Ngouabi University B.P. 69, Brazzaville (Congo)

Abstract: Strychnos camptoneura is an essential ethnomedicinal plant of the Congolese traditional medicine known to prevent, mitigate or treat the skin diseases. We aimed to investigate the in vitro anti-inflammatory effects of the S. camptoneura seeds ethanolic extract (EE), using the human keratinocytes and dermal fibroblast cells, and the in vivo immuno-modulatory effects. The in vitro anti-inflammatory activity was evaluated using the lipopolysaccharide-induced inflammation model. For the in vivo immuno-modulatory activities of ethanolic extract, immune cells were quantified by flow cytometer technique. The results showed that EE exhibited a higher anti-inflammatory activity compared to a non-treated group. The immuno-modulatory assay revealed that EE stimulate the immune cells (CD3, NK cells) and circulating anti-inflammatory cytokines (IL-10 and IL-4). These results could constitute a scientific basis for using S. camptoneura in traditional medicine.

Keywords: Strychnos camptoneura, cytotoxicity, anti-inflammatory, immuno-modulatory, human cells, immune cells

Received October 10, 2022; Revised November 17, 2022; Accepted November 25, 2022

Citation: Morabandza Cyr Jonas, Moulari Brice, Gombé Assoungou Hermann, Abena Ange Antoine, In vitro Anti-inflammatory and in vivo Immuno-modulatory Activities of Ethanolic Extract of Strychnos camptoneura (Loganiaceae) Seeds. American Journal of Pharmacological Sciences, 10 (1). 38-46. January 2022. doi:10.12691/ajps-10-1-7

1. Introduction

In recent years, inflammation responses with Celsus’ four cardinal signs, namely heat, pain, redness and swelling have attracted increasing attention [1]. Inflammation responses play an important role in multiple diseases with a high prevalence among population, such as hepatitis [2], lung disease [3] and Alzheimer’s disease [4]. And, they are also centrally related to the pathogenesis of a large number of acute and chronic diseases, such as rheumatoid arthritis [5], colonic inflammatory response [6], dermatitis [7]. However, the conventional therapies for inflammation, including steroids and nonsteroid anti-inflammatory drugs (NSAID) [8,9,10], have shown many side effects and deficiencies. Considering this, Strychnos camptoneura is an essential ethnomedicinal plant of the Congolese, which its use in traditional medicine is known for its therapeutic ingredients to prevent, mitigate or treat many diseases. Traditionally various parts of Strychnos camptoneura such as barks, leaves and seeds of this plant are used for the treatment of various skin infections, inflammatory, pains, burns… [11]. According to recent reports, Strychnos camptoneura is a polyphenol-rich plant [12]. Numerous studies have attributed to polyphenols a broad range of biological activities including but not limited to anti-inflammatory, immune-modulatory, antioxidant, cardiovascular protective and anti-cancer actions [13,14,15,16,17]. Polyphenols are ubiquitously made by plants and are present either as glycosides esters or as free aglycones [18]. More than 8000 structural variants exist in the polyphenol family. Polyphenols are bioactive compounds found in fruits and vegetables contributing to their color, flavor, and pharmacological activities [13]. They are classified according to their chemical structures into flavonoids such as flavones, flavonols, isoflavones, neo-flavonoids, chalcones, anthocyanidins, and pro-anthocyanidins and non-flavonoids, such as phenolic acids, stilbenoids, and phenolic amides [19]. Also, many epidemiological and experimental research have been studying the anti-inflammatory and immune modulation activities of polyphenols [20,21]. The ability of these natural compounds to modify the expression of several pro-inflammatory genes like multiple cytokines, lipoxygenase, nitric oxide synthases cyclooxygenase, in addition to their antioxidant characteristics such as ROS (reactive oxygen species) scavenging contributes to the regulation of inflammatory signaling [22,23]. Also, we know that in inflammatory and immune reactions, many immune cells play an important role. For example, macrophages play critical roles, they protect body from external intruders through phagocytosis in producing many inflammatory mediators such as TNFα and IL-1β [24,25]. On the other hand, LPS, a component of the Gram-negative bacteria cell wall, has been often used in inflammatory response because it can activate several immune cells and intracellular signaling pathways, including NF-kB and MAPK pathways [26,27]. The present study examined the potential for seeds ethanolic extract of Strychnos camptoneura to reduce inflammation effects in LPS-stimulated human keratinocytes and fibroblasts in vitro. Also, its immunomodulatory effect has been examined in vivo.

2. Material and Methods

2.1. Plant Material and Preparation of the Extract

Seed samples of Strychnos camptoneura (Loganiaceae) (S. camptoneura) used in this study were collected from the western-Cuvette department in the Itoumbi district (Congo-Brazzaville) in July 2019. These samples were identified by reference to the herbarium of the Exact and Natural Sciences Research Institute of Congo (IRSEN). Voucher specimens are preserved at the herbarium of IRSEN under the number 271. The S. camptoneura seeds were dried at room temperature and stored until the extraction procedure. Briefly, the dried seeds (100 g) were extracted by the maceration technique, placed in 90% ethanol under magnetic stirring for 72 hours. The solutions obtained were filtered three (3) times with the absorbent cotton and the filter paper, concentrated under reduced pressure (BÜCCHI rotavapor) and then preserved at +4 °C until in vitro tests.

2.2. Quantitative Phytochemical Screening

Preliminary phytochemical screening of the S. camptoneura seeds ethanolic extract (EE) was tested for alkaloid, terpenoid, flavonoid, tannin, reducing sugars and saponin. The results are indicated as (+) for the presence and (−) for the absence of phytochemicals.

2.2.1. Test for Alkaloid

A few drops of Dragendroff’s were applied to a test tube of about 1 ml of extract and color change was detected. The occurrence of orange color was a sign of the presence of alkaloids [28].

2.2.2. Test for Terpenoid

About 5 ml of plant extract was applied to 3 ml of chloroform and 2 ml of concentrated sulphuric acid (H2SO4). The presence of terpenoids was observed by reddish-brown color [29].

2.2.3. Test for Flavonoids

In this test, 2 ml of plant extract was treated with 5 drops of diluted sodium hydroxide (NaOH), followed by diluted hydrochloric acid (HCl). A yellow solution with NaOH turned colorless with dilute HCl indicated the existence of flavonoids [30].

2.2.4. Test for Tannin

About 2 ml of tested plant extract was stirred with 3 ml of distilled water and five drops of ferric chloride (FeCl3) were added. The formation of dark blue precipitate was the indication of tannins [31].

2.2.5. Test for Saponin

In this test, about 5 ml of tested extract was shaken with 5 ml of distilled water in a test tube. The formation of foam was considered as an indication of the presence of saponins [32]. The reducing sugars were characterized by Fehling’s test.

2.3. Cell Culture and in vitro EE Cytotoxicity Experiment

Two cellular types were used in this study including two human lineages HaCat keratinocytes (CLS 300493) and the skin fibroblasts (ATCC CRL-2522). These cells were purchased from American Type Culture Collection (ATCC-Manassas, USA). The cells were plated in 96-well culture plates (Corning Inc., Corning, NY, USA) at 1x10⁴ cells/well in DMEM medium (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution, the 50 ml cell culture flask was used. Cells were cultivated in an incubator at 37°C with 5% carbon dioxide. After the formation of a monolayer, the plate was gently centrifuged at 150 g (Beckman, Indianapolis, IN, USA) for 5 min. Then, the supernatant was gently removed and replaced with 200 μL of cells culture medium containing different concentrations of the ethanolic extract (1.56, 15.12, 31.25, 62.5, 125, 250 μg/mL). After 24 h of incubation, cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, Hamburg, Germany) colorimetric assay described by Mosmann [33]. Absorbance was measured at 570 nm with a spectrophotometer (Varioskan, Thermo Scientific, Saint-Herbain, France). Cytotoxicity was expressed as a percentage of controls (untreated cells), and the IC50 values of EE were calculated for each cell line used.

2.3.1. Effect of Lipopolysaccharide (LPS) on Cells

The inflammatory cell model used in this study is the one based on lipopolysaccharide (LPS) described by many authors [34,35]. In order to determine the non-cytotoxic concentration of LPS which induces the maximum pro-inflammatory cytokine, a cytotoxicity of LPS on cell lines was assessment. For these assays, cells were seeded on 96-well microplates(Becton Dickinson, Meylan France) at a density of 1 × 10⁴ cells per well. The cells were fed every two days with culture medium and used day 7 after seeding. Briefly, the medium was removed, cells were washed with Hank's Balanced Salt Solution (HBSS) (Gibco, France) twice, and the LPS (100 μL) at different concentrations were added to the cells. Microplates were incubated at 37 °C to evaluate a cellular viability for 4 h. Then, cell viability was assessed using the MTT colorimetric assay. The cell supernatants were used to quantify the pro-inflammatory cytokines.

2.3.2. Investigation of the Effect of EE on Pro-inflammatory Cytokines Activation by LPS

The cell lines were grown in DMEM supplemented with FBS and antibiotics as described above. The cells were seeded in 96-well plates at a density of 2 × 10⁴ cells per well and were allowed to adhere for 24 h. 10 μg/ml of lipopolysaccharide (LPS) was added to one part of the cells to induce cell inflammation (pro-inflammatory cytokines secretion). After 4 h the cells were washed twice with DPBS (Dulbecco's phosphate-buffered saline) (Gibco, France), fresh medium was added, and the cells were (or no) treated with EE at 0.75 and 1.56 μg/ml. After incubation for 8 h, 24 h and 48 h cell supernatants were collected, centrifuged and analyzed for the amount of TNFα and IL-1β with commercial ELISA kits (Thermo Scientific, Saint-Herbain, France).

2.4. In vivo immuno-modulatory Effect of EE

4–6-week-old male Swiss/CD-1 mice (average weight 25 g) were purchased from Janvier Labs (Saint Berthevin, France). The animals were kept at room temperature (25 ± 2 °C) and relative humidity (40–60%) under a 12 h light/dark cycle. Food and water were provided ad libitum. All studies were approved by the Institutional Animal Care and Use Committee of the University of Franche-Comté (experimentation authorization number A-25-48) and were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, USA). All animals were divided into 3 groups, each consisting of 6 mice. One group was chosen as the baseline (negative control), the second group was treated with ethanolic extract at dose of 150 mgkg⁻¹ of body weight of mice and the last group was used as a positive control group treated with beta-1,3-glucan at 150 mgKg⁻¹ body weight ((Sigma-Aldrich, Hamburg, Germany).After acclimatation period, mice were received via oral gavage the different formulations on one day. The negative control group was administered with sterile phosphate-buffered saline (PBS). 24 h after gavage, blood was collected via caudal vein using the sodium citrate as anticoagulant for the immune assays.

2.4.1. Isolation and Analysis of Blood Immune cells

Staining of cellular surface marker was performed using freshly collected whole blood. Briefly, two antibody staining cocktails composed of anti-mouse CD45-PE-Texas-Red, CD11b-FITC, CD11c-PE-Cy7, F4/80-APC (cocktail 1) and CD45-PE-Texas-Red, CD3-BV421, NK-APC, CD4-Horizon V500, CD8-FITC (cocktail2) as specific markers of the myeloid lineage and lymphocytes lineage, respectively. These antibodies were purchased from Thermo Scientific (Saint-Herbain, France).Then, the following mixture was realized: 1.25 µl or 25 µl of blood sample and 50 µl of cocktail 1 or 2 were incubated on ice and in the dark. Following 15 min incubation, 350 µl of Red Blood Cell Lysing Buffer 1X were added and incubated during 20 min. after the last incubation 100 μl of each cell suspension was placed in polypropylene tubes and the Flow cytometric analysis of cell subtypes was performed using BD Canto II flow cytometer (BD Bioscience), and the results were analyzed using the FACS DIVA software.

2.4.2. Circulating Anti-inflammatory Cytokines

To assess the ethanolic seeds extract effect on circulating anti-inflammatory cytokines, mice (n = 6/group) were treated with either EE (orally) for 1 day. Blood samples were taken 24 h after the treatment and analyzed using IL-4/IL-10 ELISA (mouse Cytokine/chemokine immunoassay, Millipore Corporation, MA, USA) according to the manufacturer’s instructions.

2.5. Statistical Analysis

The results were expressed as mean values ± S.D. For the analysis of statistical significance ANOVA followed by Dunn's test for all pairwise comparison in case of multiple comparison were applied, excepted when normality and equal variance were passed, it was followed by the Tukey test. Student's t test was applied to study the significance of difference between two treatment groups, however, if normality and/or equal variance was not achieved the Mann–Whitney U test was applied. In all cases, p < 0.05 was considered to be significant.

3. Results

3.1. Qualitative Phytochemical Screening

The phytochemical screening of S. camptoneura seeds extract revealed the presence of main categories of phytochemical compounds which are terpenoid, flavonoid, and carbohydrates (reducing sugars) as shown in Table 1.

Table 1. Phytochemical screening of S. camptoneura seeds ethanolic extract

3.2. Cytotoxicity of EE

To address the in vitro toxicity of the ethanolic extract of S. camptoneura, human HaCaT keratinocytes and skin fibroblasts cells were cultivated with different concentrations of the extract as described above.

Regardless the concentrations varying between 0 and 1.56 µg/ml, we observed the cell viability superior at 70% and 80% for keratinocytes and dermal fibroblasts respectively. And for a concentration superior at 1.56 µg/ml, the cell viability was inferior at 70% and 80% (Figure 1A and Figure 1B). Cell viability was highest with dermal fibroblast cells compared to keratinocytes. IC₅₀ values of EE were 60.26 ± 1.0 µg/ml and 296.08 ± 11.2 µg/ml for keratinocytes and fibroblast cells respectively (Figure 1C). Thus, in the remainder of this assessment, only the concentrations giving at least 70% and 80% cell viability for keratinocytes and fibroblasts respectively, will be used (0.75 and 1.56 µg/ml).

Figure 1. Seeds ethanolic extract of S. camptoneura (EE) solutions of different concentrations were tested for their cytotoxicity to HaCaT cells (A), dermal fibroblast cells (B) after incubation for 8 h. IC₅₀ values were determined using the graph (C). Data are shown as mean±S.D

3.3. In vitro Inflammation Model Induced by LPS

Figure 2A and Figure 2B show the cytotoxicity effect of LPS on cells at different concentrations. We observed the no-alteration in cell viability for all concentrations tested, the cell viability was superior or equal at 90%. TNFα secretion from non-stimulated cells was slightly increased. However highest levels were reached with stimulated cells (Figure 2C). Similar tendencies were observed with IL-1β (Figure 2D).

Figure 2. LPS solutions of different concentrations were tested for their cytotoxicity to HaCaT cells (A), dermal fibroblast cells (B) after incubation for 8 h. (C) and (D) represent the TNFα and IL-1β secretion after cell activation during 4 h by LPS. Data are shown as mean±S.D. *p<0.05 compared with non-activated cells (concentration 0)

3.4. In vitro Anti-inflammatory Effects of EE

TNFα and IL-1β secretions from non-stimulated cells (healthy controls) were slightly increased for all concentrations. However highest levels were reached with cells activated by LPS. But on cells-activated by LPS and treated with seeds ethanolic extract of S. camptoneura (EE), TNFα and IL-1β beta secretion decrease depending on the incubation time (Figure 3). At 8 h incubation, all ethanolic extract concentrations significantly decrease the secretion of both cytokines from human HaCaT and fibroblast cells. Similar tendencies were observed at 24 and 48 h (Figure 3A, B, C, D). Also, we noted that alone TNFα secretion gives statistically significative difference between both tested concentrations of EE (Figure 3A, Figure 3B).

Figure 3. TNFα (A, B) and IL-1β (C, D) secretions of non-activated (healthy) and activated keratinocytes and fibroblast cells after treatment with different concentrations EE at different incubation time; data given as mean±S.D., *p<0.05 compared with LPS control; *p<0.05 compared with EE at 0.75 μg/ml

3.5. In vivo Immunomodulatory Effect of EE

To assess the in vivo immunomodulatory effect of EE, we quantified circulating anti-inflammatory cytokines (IL-4 and IL-10), myeloid and lymphocytes cells after EE oral administration in mice. Cytokines were determined by ELISA assay technique; myeloid and lymphocyte cells were determined by Fluorescence activated cell sorting (FACS). Results obtained show these markers secretion-increasing after mice treatment of EE. The following secretion levels were obtained: IL-10 (non-treated group: 41.27±6.86 pg/ml; EE-treated group: 80.93±8.37 pg/ml), IL-4 (non-treated group: 16.20±6.41 pg/ml; EE-treated group: 30.39±2.60pg/ml), CD4 (non-treated group: 75.03±1.55 %; EE-treated group: 79.30±1.40 %; beta-1,3-glucan-treated group: 88.20±1.05 %), CD3 (non-treated group: 29.63±3.70 %; EE-treated group 37.06±4.10 %; beta-1,3-glucan-treated group: 54.86±6.73 %), NK cells (non-treated group: 13.73±3.25 %; EE-treated group: 23.13±2.35 %; beta-1,3-glucan-treated group: 27.37±4.50) (Figure 4, Figure 5 and Figure 6). We spotted a no-modulation on F4/80, CD11b/CD11c and CD8 cells when mice were treated with EE (Figure 4E, Figure 4F).

Figure 4. Flow cytometric analysis of blood immune cells in the animals of the non-treated, positive controls, and EE-treated groups in percentage. Myeloid cells were identified based on the expression of CD11b CD11c and F4/80; lymphocyte cells were identified based on the expression of CD3, CD4, CD8 and NK (natural killer). Data are shown as mean ± SD for n = 6 animals per group. *p< 0.05 compared with non-treated control, £p< 0.05 compared with EE-treated group. ANOVA followed by Dunn’s test was used for all pairwise multiple comparisons

Figure 5. pictures of Flow cytometric analysis of blood immune cells in the animals of the non-treated, positive controls, and EE-treated groups. Myeloid cells were identified based on the expression of CD11b CD11c and F4/80; lymphocyte cells were identified based on the expression of CD3, CD4, CD8 and NK (natural killer)

Figure 6. Effects of EE in circulating anti-inflammatory cytokines (IL-4 and IL-10).*p < 0.05 compared with non-treated group

4. Discussion

We noticed that study concerning the anti-inflammatory efficacy and the immunomodulation effect of S. camptoneura seeds are non-existent despite the fact that various parts of this plant are used, in traditional medicine, for the treatment of various skin diseases such as burns, infectious and inflammation. In our study, we observed significantly in vitro anti-inflammatory effect after application of ethanolic extract of S. camptoneura seeds compared to the control which was corroborated by the decrease values of pro-inflammatory characteristic markers such as TNFα and IL-1β. An in vivo immunomodulatory effect was present after the treatment by EE compared to the control, with an interessant stimulation levels of some immune cells.

We investigated the phytochemical analysis and the effects of S. camptoneura seeds ethanolic extract in cell viability, in vitro anti-inflammatory activity using the human dermal fibroblast and keratinocytes (HaCaT) cells. The LPS-induced cellular inflammation model was used. Also, its ability to induce the in vivo immunomodulation was assessed on mice.

The content of flavonoids, tannins and reducing sugar in the S. camptoneura seeds ethanolic extract confirmed the results of previous studies [12]. The presence of these compounds, and particularly flavonoids, could well contribute to the biological activity of this extract in accordance with literature data which demonstrate their important role in the anti-inflammatory process [36,37].

The cell viability shows the variable IC50 values to dermal fibroblast cells and keratinocytes suggesting that cells, which are fundamental to the cutaneous anti-inflammatory process, are differently sensitive to the effects of the ethanolic extract tested. In presence of EE, we observed a significant anti-inflammatory effect in fibroblast and keratinocytes cells, with an important decrease of pro-inflammatory markers (TNFα and IL-1β).

It is known that a delicate balance between pro-inflammatory and anti-inflammatory immune responses is required to clear pathogens without causing autoimmunity [38]. TNFα and IL-1β are potent inflammatory cytokines that regulate immunity and disease pathogenesis [39]. Aberrant overproduction of TNFα had been implicated in the pathogenesis of chronic inflammatory diseases such as rheumatic arthritis, psoriatic lesions/arthritis, dermatitis and inflammatory bowel disease [40,41]. Similarly, elevated IL-1β expression led to dermatitis and other chronic inflammations [39,42]. Thus, TNF-α and IL-1β play an important role in inflammatory disease birth [40]. The TNFα or IL-1β down-regulation by EE could be due to the presence of flavonoids compounds in this plant as we showed. Indeed, the role of Flavonoids in inflammation treatment is well established in literature [43]. And the mitogen-activated protein kinases (MAPK) are a highly conserved family of serine/threonine protein kinases. They play a key role in a range of fundamental cellular processes like cell growth, proliferation, death and differentiation. They regulate gene transcription and transcription factor activities involved in inflammation. However, it been showed that flavonoids (polyphenols) can block TNFα release by modulating MAPK pathway at different levels of the signaling pathway. For example, Luteolin reduces TNFα liberation by LPS-activated mouse macrophages, it blocks ERK1/2 and p38phosphorylation [44]. In epithelial cells, luteolin, as well as other polyphenols such as chrysin and kaempferol block TNFα triggered ICAM-1 expression by inhibiting ERK, JNK and P38 [44,45]. Quercetin blocks the phosphorylation of ERK, JNK in THP-1 activated human monocytes, while in murine macrophages RAW 246.7 triggered by LPS it blocks the phosphorylation and the activation of JNK/SAPK (stress activated protein kinases), ERK1/2, and p38 leading to a reduction in the transcription and expression of TNFα expression [46]. EE, rich in flavonoids, could be act on MAPK signaling pathway leading to TNFα down-regulation in EE-treated groups.

In immunomodulatory effects, our results suggest that EE has proliferative effect on lymphocyte lineage cells (CD3, CD4 and NK cells). But it hasn’t proliferative effect on myeloid lineage cells (CD11b/c, F4/80). This stimulator effect of EE on lymphocyte lineage cells could be due to the presence of polysaccharides and flavonoids. Indeed, many previous works showed that plant-derived polysaccharides have proliferative effect on lymphocytes. Behravan et al. [47] reported that crude ethanolic extracts of Portulaca oleracea shoots protected against oxidative DNA damage in lymphocytes, effectively allowing them to proliferate. There have been in vivo assays using mice administered with these polysaccharides from some plants species, and the results indicated that they promote lymphocyte proliferation [48,49]. Although the effect of EE on some lymphocyte’s proliferation was not significant in this study, it is still remarkable to note that the increase in proliferation relative to the beta-1,3-glucan-treated mice (positive control) was enough similar. This is confirming then that the EE, rich in polysaccharides, contains immunoactive compounds that could induce proliferation of lymphocyte cells. Aside from the potential immunoactive role of polysaccharides that were reported to be present in S. camptoneura seeds, the present study indicates that the presence of flavonoids in the EE extract could have also produced the immunomodulatory effects in lymphocytes as shown by Kuo et al. [50]. They showed in vivo that tea extract, rich in flavonoids, increase NK cell activity thus confirming our results on NK proliferative after EE-treatment.

Finally in this study we observed that EE stimulates IL-10 and IL-4 secretion. This may be due to posttranscriptional effects of the flavonoids, since it is well established that IL-10 can be regulated by posttranscriptional mechanisms in macrophages. Based on this evidence, we suggest that EE would act positively to p38-MAPK, which is largely involved in the regulation of IL-10 and IL-4 expression as shown by Comalada et al. [51].

5. Conclusion

In conclusion, our data propounds that S. camptoneura ethanolic extract contains major compounds that, either alone or in association with the minor components present in the seeds plant extract, can trigger the cellular events, critical to anti-inflammatory and immuno-modulation effects. However, future studies need to be implemented for further elucidation on actives compounds. This study on anti-inflammatory and immuno-modulator effects of S. camptoneura seeds extract could constitute a scientific basis of using of S. camptoneura in traditional medicine.

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