Terminalia brownii Fresen: Stem Bark Dichloromethane Extract Alleviates Pyrogallol-Induced Suppression of Innate Immune Responses in Swiss Albino Mice

Terminalia brownii is widely used in folklore medicine and has diverse biological activities. However, its effect on the immune system is yet to be studied. Therefore, our study evaluated the immunomodulatory effect of T. brownii on nonspecific immunity. Innate immunity is the initial defence phase against pathogens or injuries. Dichloromethane plant extracts were tested on female Swiss albino mice and Wister rats. The effect of the extract on innate immunity was assessed via total and differential leukocyte counts, tumor necrosis factor-alpha, and nitric oxide production by mouse macrophages. The 3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide assay was employed for viability testing. Phytochemical profiling was carried out using gas chromatography-mass spectrometry, while toxicity studies were carried out following the Organization for Economic Cooperation and Development guidelines. Our results demonstrated that administration of T. brownii stem bark dichloromethane extract to pyrogallol-immuno compromised mice significantly (p < 0.05) increased total and differential leukocyte counts compared with the control. The extract showed no adverse effect on the viability of Vero cells and macrophages and significantly (p < 0.05) augmented tumor necrosis factor-alpha and nitric oxide production. Hexadecanoic acid, linoleic acid, octadecanoic acid, squalene, campesterol, stigmasterol, and β-sitosterol, all of which stimulate, were identified in the extract. The extract did not cause any death or toxic signs in rats. In conclusion, T. brownii dichloromethane extract has an immunoenhancing effect on innate immune responses and is not toxic. The observed immunoenhancing impact of the extract was attributed to the presence of the identified compounds. The results of this study provide crucial ethnopharmacological leads towards the development of novel immunomodulators for managing immune-related disorders.


Introduction
Innate immunity is the initial phase of defence against viral and bacterial infections and sterile infammation [1]. It is a nonspecifc defence mechanism that the host uses after an antigen encounter, instantly or within a few hours. However, innate immunity does not have immunologic memory. Tus, it cannot recognise the same pathogen if the body encounters it in the future [2]. Innate immunity has diferent types of protective barriers, including anatomic (mucous membrane and skin), physiologic (chemical mediators, low pH, and temperature), phagocytic and endocytic, and infammatory barriers [2]. Nonspecifc immunity comprises cellular and humoral components that identify, inactivate, and kill invading pathogens [3]. Te cellular part includes phagocytes (neutrophils and macrophages), eosinophils, basophils, monocytes, mast cells, dendritic cells, innate lymphoid cells, and natural killer cells [2,4]. However, the humoral component includes complement proteins, collectins, and antimicrobial peptides, among other elements [5]. Primary innate immune responses involve the recruitment of efector cells to the site of injury or pathogen invasion, and infammation, through the production of chemokines and cytokines. Te produced cytokines, tumor necrosis factor (TNF), interleukin 6 (IL-6), and interleukin 1 (IL-1), also trigger local cellular responses to injury or infection and fever development [2].

Plant Materials.
A stem bark sample of T. brownii tree ( Figure 1) was acquired from Kitui, Kenya (1.3099°S 37.7558°E; about 152 km from Nairobi) in May 2021, aided by a native herbalist. Botanical verifcation was conducted at the East African Herbarium situated at the National Museums of Kenya, and a voucher specimen was deposited there (accession number: JWM001). Te plant sample was taken to the animal breeding and experimentation facility at Kenyatta University for preparation and bioassays. Te sample was rinsed using running tap water, reduced into tiny shreds, and shade-dried till thoroughly dried and powdered [40].

Extraction of Plant Materials.
Extraction was carried out using dichloromethane (DCM) (Shandong Arctic Chemical Co. Ltd, Shandong, China). Tree litres of DCM were added to 1500 g of the samples' powder. An aspirator pump, VE-11; Pfeifer Vacuum, Inc., Nashua, NH, was employed to flter the extracts. Te extracts were concentrated using a RE-501 rotary evaporator (Zhengzhou Keda Machinery and Instrument Equipment Co., Ltd, China) in vacuo. Te extracts were stored at 4°C [41].

Experimental Animals. Te Kenyatta University Animal
Care and Use Committee (PKUA/005/005) authorized this study. Female Swiss albino mice (7-9 weeks old; 25-30 g) were used for immune response studies. According to [42], mice have played a signifcant role in many vital advances in immunology; hence, they are used in this study. Female mice were chosen for this study because they mount more robust immunological responses than males [43,44]. On the other hand, female Wistar rats (2-3 months old; 140-180 g) were used for toxicity studies as guided by the OECD guidelines [45,46]. Te experimental animals were purchased from the animal breeding and experimentation facility at Kenyatta University. Tey were nourished with standard rodent pellets (Specialty feeds, Glen Forrest, West Australia) and water ad libitum. Te animals were allowed to acclimate to laboratory conditions for a week before the bioassay tests. Te composition of the standard rodent pellets is shown in Table 1 [47].

Evaluation of the Immunomodulatory Activity of T. brownii Stem Bark DCM Extract.
Te immunomodulatory activity of T. brownii DCM extract was assessed by evaluating total and diferential leukocyte counts, nitric  . Te outfows were centrifuged at 1000 rpm for 20 min, and RPMI 1640 culture medium was employed to rinse the cells twice. RPMI 1640 medium enhanced with L-glutamine (2 mM), gentamicin (0.04 mg/ml), 10% newborn calf serum, and penicillin 100 μ/ ml were used to resuspend the cells [49]. Cell viability was assessed using methylene blue assay; viability was found to be above 95%.
(2) Efect of T. brownii Stem Bark DCM Extract on Vero Cells and Macrophage Viability. Viability tests were conducted via the MTT assay using 96 well culture plates [50]. Vero cells were procured from the American Type Culture Collection (ATCC), Manassas, Virginia. 100ÂμL RPMI 1640 culture medium augmented with 10% fetal bovine serum was pipetted into all wells. A hundred microlitres of the extracts (3 mg/ml) were introduced into the culture plates' bottom row (row H) in triplicates separated by blanks. Levamisole (1 μg/mL) was used as a positive control in assessing TNF-α and nitric oxide production by macrophages. Serial dilutions (two-fold) of the extracts were conducted up from row H to row C, and the excess (200 μl) solution was disposed of. Rows B and A were left as the control wells. Te plates were incubated in a carbon dioxide (CO 2 ) incubator for 24 h. After incubation, supernatants were drawn out to assess NO and TNF-α production by macrophages. Forty microlitres of MTT were added to all wells, and the plates were incubated for four hours. Culture media and MTT were pipetted out of the wells, DMSO (100 μl) was pipetted into the wells, and plates were incubated further for 20 min. Enzyme-linked immunosorbent assay (ELISA) reader (Biobase Industry, China) was used to read the plates at 570 nm. Te following formula was used to compute the percentage viability: Cell viability � Sample's or standard drug's absorbance Control's absorbance * 100. (1) (3) Evaluation of NO Production. Te Griess reagent system was employed to estimate the levels of the NO produced by macrophages [51]. 50 μl of the supernatants were admixed with 50 μl Griess reagent in 96 well plates and incubated at room temperature for 10 min. An ELISA reader (Biobase Industry, China) was used to obtain absorbance values at 570 nm. A 0-100 μM nitrite standard reference curve was generated and used to assess the concentration of nitrites.

Efect of T. brownii Stem Bark DCM Extract on the Production of TNF-α by Mouse Macrophages.
Mouse-specifc T1/T2 cytokine Cytometric Bead Array (CBA) kit (BD Biosciences, US) was used to determine levels of TNF-α in macrophage culture supernatants as directed by the manufacturer. 50ÂμL of bead populations with distinctive fuorescent extents, conjugated with analyte-specifc antibodies, were added to 50ÂμL of the samples and incubated for 90 min at room temperature. A standard curve was generated for the analyte. Samples and standards were rinsed to remove any detached components. Phycoerythrinlayered detection antibodies were added, incubated for an hour, and rinsed of. Fluorescence-activated single cell sorting (FACS)-Calibur fow cytometer (BD Biosciences, US) was used to quantitate the fuorescent signals generated. A Flow Cytometric Analysis Program (FCAP) software (BD Bioscience, US) was employed to establish the concentration of the analyte [52,53].

Acute Toxicity
Study. An acute toxicity study was conducted as outlined in the OECD guidelines for the acute oral toxicity up-and-down procedure [46]. A limit test was Evidence-Based Complementary and Alternative Medicine performed since aqueous and methanol extracts of T. brownii bark were previously reported to cause no death or toxicity at 2,000 mg/kg BW dosage in mice [39]. Female Wistar rats were divided into normal and test groups. A gavage needle was used to give the treatments as a single dose. Rats were fasted overnight, weighed, and treated. Te animals further fasted for four hours. Te control group received 2.5% DMSO in distilled water, while the test group received stem bark DCM extract of T. brownii at 2,000 mg/kg BW. Rats were observed for death and toxicity signs, including lethargy, coma, sleep, diarrhea, salivation, convulsions, tremors, and changes in the skin, fur, eyes, and mucous membrane. Te body weights of the rats were retaken on days 7 and 14. After the study period, the animals were placed in a desiccator containing diethyl ether, and after they died, their carcases were incinerated.

Subacute Toxicity Study.
Tis study followed the OECD guidelines for the repeated dose 28-day oral toxicity study in rodents [45]. Female Wistar rats were categorised into four groups. Te frst group was the control and received 2.5% DMSO in distilled water. Te second, third, and fourth groups received T. brownii stem bark DCM extract at 300, 520, and 900 mg/kg BW dosages, respectively. A gavage needle was used to administer the treatments daily for 28 days. Rats were observed for death or toxicity signs daily; body weights were taken and recorded weekly. On day 29, the animals were weighed and sacrifced. Blood was drawn through cardiac puncture for haematology and biochemistry analysis. Body organs were also harvested, and their weights were taken.

Analysis of Biochemical and Hematological Parameters.
Blood samples for biochemistry analysis were put in Eppendorf tubes, while samples for haematology analysis were put in ethylenediaminetetraacetic acid (EDTA) tubes. Te blood was centrifuged for 10 min at 3000 rotations per minute to acquire serum for biochemistry analysis [54]. Parameters assessed in both biochemistry and haematology are as listed by OECD guidelines [45].

Phytochemical Profling of T. brownii Stem Bark DCM
Extract. Gas chromatography-mass spectrometry (GC-MS) analysis of the plant extracts was carried out as described by [55] with a few modifcations. A hundred milligrams of the sample were weighed, and 1 mL DCM was added. Te mixture was vortexed for 10 s, sonicated for 10 min, and centrifuged for 5 min at 14,000 rpm. Te supernatant was fltered and diluted to prepare 100 ng/μL. Analysis was carried out on four replicates. Te GC-MS instrumentation included a TRACE GC Ultra Gas Chromatograph conjoined with a Termo mass spectrometer detector. Te GC-MS system was ftted with a TR-5 MS column. Helium was used as the carrier gas and maintained at a fow rate of 1.0 ml/min and a 1 : 10 split ratio. Mass spectra were acquired via electron ionization (70 eV), using an m/z 40-450 spectral range. Quantifcation of the compounds was by the metabolites as distinguished by the mass spectrometer.
Te detected compounds were identifed using the AMDIS software by the retention times and mass spectrum analogous to standards (where available), and the National Institute of Standards and Technology (NSIT) database. Comparison with spectra of known compounds in the literature was also used to identify the detected compounds.

Data
Analysis. GraphPad Prism 8 statistical software was used for data analysis. One-way analysis of variance (ANOVA) was carried out to assess the diferences between groups. For mean values separation, Tukey's post hoc test was conducted. Values at p < 0.05 were considered statistically signifcant.

Sample Powder and Plant Extract
Yield. Four kilograms of fresh stem bark sample of T. brownii yielded 2.95 kgs of sample powder. Dichloromethane produced 28 g extract from 1500 g of T. brownii stem bark powder sample.

Efect of T. brownii Stem Bark DCM Extract on Total and
Diferential Leucocyte Counts. Terminalia brownii DCM extract enhanced the innate immunity of pyrogallolimmunosuppressed mice. Te extract signifcantly increased total white blood cells (WBCs), neutrophils, lymphocytes, monocytes, eosinophils, and basophils count in the extract-treated mice compared with the negative control group (p < 0.05) ( Table 2). Te extract at 50, 100, and 150 mg/kg BW dosages produced a dose-dependent response in the increase of neutrophils. However, the efect of the extract at dosages 50 and 100 mg/kg BW on total WBCs, lymphocytes, eosinophils, and basophils was comparable (p > 0.05). Te standard drug, levamisole, demonstrated a signifcantly (p < 0.05) higher activity in the augmentation of leukocyte counts compared to the extract at a dosage of 150 mg/kg BW (Table 2).

Efect of T. brownii Stem Bark DCM Extract on the Viability of Vero Cells and Macrophages. Terminalia brownii
DCM extract showed no toxicity towards Vero cells; the extract at the highest dosage resulted in viability beyond 50% (Table 3). Te extract produced a dose-dependent response at the tested concentrations (Table 3), and there was a signifcant (p < 0.05) diference between the activity of the extract at all concentrations and the control (Table 3).
Te dichloromethane extract of T. brownii was not toxic to macrophages, as shown by the above 50% viability produced by its highest concentration (Table 3). Te extract had a signifcant (p < 0.05) diferent efect on macrophage viability from dosage 187.5 μg/ml to 3000 μg/ml (Table 3). Furthermore, compared to the control, DCM extract at all tested doses had a signifcantly diferent efect on the viability of macrophages (p < 0.05) ( Table 3).

Efect of T. brownii Stem Bark DCM Extract on NO
Production by Mouse Macrophages. Te dichloromethane extract of T. brownii augmented NO production by macrophages obtained from pyrogallol-immunosuppressed mice. Te extract, from dosage 187.5 μg/ml to 3000 μg/ml, produced a signifcant (p < 0.05) diference in the production of NO compared to the untreated cells (Table 4). Terminalia brownii DCM extract at 375, 750, 1500, and 3000 μg/ml dosages demonstrated a dose-dependent response. Furthermore, there was a signifcant (p < 0.05) diference in the activity of the extract at the highest dose, 3000 μg/ml, and the standard drug (Table 4).

Efect of T. brownii Stem Bark DCM Extract on TNF-α Production by Mouse Macrophages.
Dichloromethane extract of T. brownii increased the production of TNF-α by murine peritoneal macrophages acquired from pyrogallolimmunocompromised mice. Te extract, at the tested concentrations, produced signifcantly (p < 0.05) elevated levels of TNF-α compared with the control (Table 5). Moreover, there was a signifcant (p < 0.05) diference between the activity of the extract at the highest dosage compared to the standard drug (Table 5). Te extract at 187.5, 375, 750, 1500, and 3000 μg/ml dosages produced a dose-dependent increment in TNF-α levels ( Table 5).

Efect of T. brownii Stem Bark DCM Extract on the Body Weights of Female Wistar Rats. Terminalia brownii
DCM extract did not afect the body weights of the experimental rats throughout the study period; extract-treated rats had comparable (p > 0.05) body weights with the control ( Table 6).

Efect of T. brownii Stem Bark DCM Extract on the Behaviour and Overall Appearance of Female Wistar Rats.
Terminalia brownii DCM extract did not afect the behaviour and general appearance of the experimental animals (Table 7).    Evidence-Based Complementary and Alternative Medicine bark DCM extract, at all tested dosages, did not afect the body weights of the experimental animals; extract-treated rats had comparable (p > 0.05) body weights with the control from weeks one to four (Table 8).

Efect of T. brownii Stem Bark DCM Extract on Female
Wistar Rats' Organ Weights. Stem bark DCM extract of T. brownii, at all tested dosages, did not cause changes in the organ weights of the rats. Relative organ weights of extracttreated rats were comparable (p > 0.05) to those of the normal control group (Table 9).

Efect of T. brownii Stem Bark DCM Extract on Female
Wistar Rats' Hematological Parameters. Dichloromethane extract of T. brownii stem bark, at all tested dosages, did not alter levels of the assessed hematological parameters; levels of the parameters were similar (p > 0.05) between the extract-treated animals and the control (Table 10).

Efect of T. brownii Stem Bark DCM Extract on Female Wistar Rats' Biochemical Parameters.
Te extract, at all tested dosages, did not afect levels of the analyzed biochemical parameters; levels of the parameters were similar (p > 0.05) between extract-treated rats and the control (Table 11).

Discussion
Our study evaluated the immunomodulatory efect of T. brownii stem bark DCM extract on innate immune responses by assessing leukocyte counts, TNF-α, and NO production by macrophages harvested from pyrogallolimmunocompromised mice. Te fndings of our study demonstrate the ability of stem bark DCM extract of T. brownii to revert immunosuppression of innate immune responses caused by pyrogallol. Te extract augmented total and diferential leukocyte counts, TNF-α, and NO production by mouse macrophages compared with the control. Tese results demonstrate the ability of T. brownii stem bark DCM extract to stimulate innate immunity, making T. brownii a potent candidate for developing new immunostimulators that can be used to manage immune-related conditions. Treatment of mice with pyrogallol resulted in the production of signifcantly lower numbers of total and diferential leukocytes compared with the control. However, stem bark DCM extract of T. brownii inversed this reduction and augmented total and diferential leukocyte counts in the blood of extract-treated mice. Leukocytes are vital cells in innate immune responses. Tey protect the body through various mechanisms, including the release of cytokines, phagocytosis, and the presentation of antigens to T cells [56]. Terefore, reduced numbers of leukocytes in the body lead to opportunistic infections since the body is not well equipped to fght invading pathogens. Hence, it is critical to maintain leukocyte counts within normal ranges to counter the immunosuppressive status [57]. Te increased leukocyte numbers in extract-treated mice could be attributed to the   Values were expressed as mean ± SEM. Statistical comparison was made along rows, and values were not signifcantly distinct by one-way ANOVA (p > 0.05). TD: T. brownii DCM extract.   Evidence-Based Complementary and Alternative Medicine capacity of the extract to induce the production of WBCs in the bone marrow or augment the production of leukocytosis mediators [58]. Te compounds identifed in the stem bark DCM extract of T. brownii augment diferent elements of nonspecifc immunity. Among the compounds identifed in the extract, only squalene and β-sitosterol enhance leukocyte count in rodents. Squalene signifcantly increased the previously reduced leukocyte counts in 3-methylcholanthrene-intoxicated rats [59]. β-sitosterol administered to mice signifcantly augmented lymphocyte counts compared to the control [60]. Te ability of T. brownii stem bark DCM extract to increase leukocyte counts in immunocompromised mice can therefore be attributed to squalene and β-sitosterol.
Te results of our study demonstrated that immunosuppression impaired the ability of mouse macrophages to produce a critical proinfammatory mediator, NO. However, stem bark DCM extract of T. brownii stimulated macrophages to produce higher NO levels. Tis result shows the ability of T. brownii DCM extract to elicit innate immune responses, especially in immunocompromised subjects, by classically activating macrophages [61]. Stimuli, including fungal and bacterial components, chemical mediators, cytokines, and bioactive plant compounds, can classically activate macrophages to make and release cytotoxic mediators, including NO and proinfammatory cytokines that help eliminate invading pathogens [51,61].
We attributed the capacity of T. brownii DCM extract to augment the NO production by mouse macrophages to the presence of hexadecanoic acid, octadecanoic acid, linoleic acid, and stigmasterol in the extract. Tese compounds increase NO production by macrophage cell lines. Low doses, 1-10 μM, of hexadecanoic acid, octadecanoic acid, and linoleic acid enhanced NO production by J774 cells [62]. Stigmasterol-treated RAW264.7 cells produced elevated levels of NO compared to the control [63]. Tis study revealed the capacity of T. brownii stems bark DCM extract to elevate TNF-α levels in macrophages isolated from pyrogallol-immunosuppressed mice. Tis fnding shows the immunostimulatory efect of the extract on innate immune responses via polarizing macrophages into classically-activated macrophages (M1 macrophages) [61]. TNF-α is a proinfammatory cytokine involved in the initiation of the expression of adhesion molecules on neutrophils and endothelial cells, leukocyte activation, and leukocyte chemotaxis [64].
Te observed capacity of T. brownii DCM extract to augment the production of TNF-α by macrophages was attributed to the presence of hexadecanoic acid, octadecanoic acid, β-sitosterol, campesterol, and stigmasterol in the extract. Tese compounds augment TNF-α production in macrophages. Hexadecanoic acid and octadecanoic acid signifcantly enhanced the production of TNF-α by J774 cells [65]. β-sitosterol signifcantly increased TNF-α production by THP-1 macrophages [66]. Hexadecanoic acid was found to activate J774A.1 macrophages classically; it signifcantly augmented TNF-α levels [67]. Te treatment of murine peritoneal macrophages with campesterol resulted in elevated levels of TNF-α compared with the control [68]. RAW264.7 cells treated with stigmasterol produced elevated levels of TNF-α in contrast to the untreated cells [63].
Our toxicity studies showed that T. brownii DCM extract is safe since it did not cause death or toxicity signs in rats. Te acute toxicity of the extract was tested at 2000 mg/kg BW. Since it did not cause any harmful efect in rats, the LD 50 of the extract was concluded to be above 2000 mg/kg, which is a wide safety margin [69]. Subacute toxicity study results further highlight the safety of T. brownii stem bark DCM extract.

Conclusions
Terminalia brownii stem bark DCM extract stimulated nonspecifc immunity in pyrogallol-immunosuppressed mice by augmenting the production of three key elements of innate immunity; leukocytes, NO, and TNF-α. Our fndings denote that T. brownii is a prospective candidate for developing new immunoenhancing medication for managing immunosuppressive ailments. GC-MS analysis of T. brownii stem bark DCM extract resulted in the identifcation of compounds shown to augment the production of leukocytes, NO, and TNF-α. Terefore, our study concludes that the observed immunostimulatory efect of the extract was due to the identifed compounds in the extract. Te DCM extract of T. brownii was safe since it did not cause any toxicity in vivo or in vitro.

Limitations of the Study
Te study conducted only quantitative phytochemical analysis of the T. brownii DCM extract; the active phytochemicals were not isolated. Furthermore, a chronic toxicity test of the extract was not conducted.

. Future Prospects
Isolation of various phytochemicals from the DCM extract of T. brownii and testing them for immunomodulatory activity. Additionally, assessment of chronic toxicity of the extract.

Data Availability
Te data supporting the authors' conclusions are available in this article.

Conflicts of Interest
Te authors declare that they have no conficts of interest regarding the publication of this article.