Evaluation of an antimalarial herbal mixture and each extract for DNA and chromosomal mutations in Swiss albino mice and Allium cepa cells

Toxicological evaluation of herbal medicines is necessary because of possible adverse effects that may be associated with their consumption. This study screened antimalarial herbal recipe (containing leaves of Azadirachta indica and stem-bark of Alstonia boonei) and its individual plant’s extract for DNA and chromosomes mutation potentials following the DNA fragmentation and Allium cepa assays. Superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) activity of the recipe and each extract was determined. The kinds of phytochemicals present in them were determined using the FTIR technique. Water extracts of A. indica, and A.boonei at all the tested doses caused significantly lower DNA fragmentations than those of the controls. However, at 25.0% and 50.0% recipe, there was no significant difference in the percentage fragmented DNA compared to the positive control (0.05% sodium azide). Cell division was significantly inhibited by the extracts and recipe, chromosomal aberrations were not dose dependently induced and were significantly lowered than that caused by sodium azide (positive control). The individual extracts and their recipe significantly inhibited Root growth. However, 12.5% recipe promoted root growth that was not significantly different from that of distilled water (negative control). SOD and CAT activities of each of the extracts and their recipe were dose dependent and significantly higher than those of the controls. Water extract of A. indica significantly suppressed generation of malondialdehyde compared to water extract of A. boonei and recipe as well as the control. The individual extracts and their recipe contained phenolic phytochemicals. The obtained results show that extract of A. indica, A. boonei and their recipe have good antioxidant properties with strong mitodepressive and root growth inhibitory effects except at 12.5% recipe. However, A. indica extract seems to have least cyto-muta-genotoxic effects than water extract of A. boonei and the recipe in mice and A. cepa cells.


Introduction
The use of plants with medicinal properties either singly (monoherb) or in combination with other plants materials (polyherbs) or non-plant materials to prevent and treat diseases is a long time practice of about 60, 000 years ago. Today, natural products and plants having therapeutic values are increasingly been sought for throughout the world and have become important sources of orthodox medicines because they are rich in bioactive phytochemicals (Haidan et al., 2016;Olorunnisola et al., 2021;Garget al., 2021). Over 50% of orthodox medicines is derived from plants. In the tropical and sub-tropical parts of the world, malaria is widely spread and it affects about 1 million people worldwide yearly, out of which 700,000 are children (Omoya and Oyebola, 2019). About 30 -50% and 25% infant morbidity and mortality, respectively are caused by malaria in Nigeria. The resistance to synthetic antimalarial orthodox drugs necessitates the increased use of herbal medicines, coupled with readily availability of medicinal plants most often with no or very low cost. Azadirachta indica (Juss) and Alstonia boonei (De Wild) belonging to the families Meliaceae and Apocynaceae, respectively, are the foremost medicinal plants employed for treatment of malaria and some other diseases in Nigeria. Their ethnopharmacological importance is perhaps based on their richness in phytochemicals with antimalarial properties. The leaf extract of A. indica contains phytochemicals such as flavonoids, glycosides, limonoids, coumarins and sterols which have schizontocidal and gametocidal effects (Afolabi et al., 2021). The stem-bark of A. boonei is known to contain phytochemicals that have therapeutic properties such as antimalarial, analgesic, antidiabetic, antimicrobial, antirheumatic and anti-inflammatory (Adotey et al., 2012). Herbal medicines preparations can be made from either one part of these plants or parts of the two plants can be combined to form a recipe. Traditionally, herbal medicines are usually prepared by combining more than one plant part to make recipe which is believed to have diverse therapeutic effects because such contains numerous therapeutic phytochemicals. The use of herbal medicines to prevent and treat diseases should be done with some levels of toxicological consciousness due to the fact that some plants extracts are associated with toxicity. Plant extracts and herbal products have been reported to have mutagenic, genotoxic, carcinogenic teratogenic effects (Akinboro and Bakare, 2007;Akinboro et al., 2011aAkinboro et al., , 2011bSponchiado et al., 2016;Akinboro et al.,2017;Babamale et al., 2017;Akinboro et al., 2020b). Herbal medicines prepared with more than one plant should always be subjected to genetic toxicological assays to establish that both the individual plant extract and the recipe itself (mixture of extracts) are safe for consumption. This study therefore aimed at investigating possible effects of individual leaves extract of Azadirachta indica and stem-bark extract of Alstonia boonei and their recipe (mixture of the two extracts) as a commonly used antimalarial herbal medicine on cell division, cellular DNA integrity and chromosome structure in plant and animal test organisms.

Plant collection and identification
Fresh leaves of Azadirachta indica (A. juss) and stem -barks of Alstonia boonei (Di wild) were respectively collected from the botanical garden, and around the New Biology Laboratory, Ladoke Akintola University of Technology (LAUTECH), Ogbomoso, Oyo State, Nigeria. They were duly identified and assigned voucher number LHO 582 and LHO 583 to Azadirachta indica and Alstonia boonei, respectively by a taxonomist, Professor A.T.J. Ogunkunle at the herbarium unit of Department of Pure and Applied Biology, LAUTECH, Ogbomoso, Nigeria.

Preparation of plant extracts
The collected plant parts (leaves of A. indica and stembark of A. boonei) were washed with clean water, thereafter air dried, before they were individually ground using 'Wing electric blender'(Malaysia). One hundred grams of the powder of each plant part was weighed on a Mettler weighing balance, while the recipe contained 50 g each of A. indica and A. boonei. The powder of each plant and that of the recipe in separate glass jars was added with five hundred milliliters of distilled water and then placed inside a water bath set at 64°C for 2 hours 15 minutes. The single extract and recipe were allowed to cool down before sieving each through a Whatman filter paper (No 1) and stored in a refrigerator at 4ºC for further use (Akinboro et al., 2017;Akinboro and Jimoh, 2021a).

Oral feeding of animals
Fifty-five female albino mice were acclimatized for 1 week at the animal house of Department of Pure and Applied Biology, LAUTECH. They were fed with commercially formulated feed and clean water ad libitum throughout the period of the experiment. The animals were weighed and grouped into 5 mice per treatment and control groups. Every three groups were administered (0.1 ml per 10 g b/w) with each of the water extract of A. indica, A. boonei and the antimalarial recipe at 25.0%, 50.0% and 100.0% doses once per day for 48 hours (Akinboro and Soremekun, 2020a). Distilled water and sodium azide (0.05%) served as the negative and positive controls, respectively.

DNA fragmentation assay
This assay was carried out spectrophotometrically as previously described by Wu et al. (2005). One gram of liver tissue from a mouse treated with each of the extract and recipe was treated in 10 ml of Tris-Ethylenediaminetetra acetic buffer. The absorbance of the reaction mixture was read at 620 nm. The percent fragmented DNA was calculated according to the formula below: ragmented DNA ( ) Absorbance of supernatant Absorbance of pellet Absorbance of supernatant

In vivo antioxidant tests 2.5.1. Lipid peroxidation (malondialdehyde generation)
The amount of thiobarbituric acid (TBA) reactive substances (TBARS) produced during lipid peroxidation was determined as previously described by Ohkawa et al. (1979). Briefly, 0.4ml of the homogenized liver tissue was mixed with 1.6ml of 0.1 MTris-KCl buffer prepared at pH 7.4. To this, 0.5 ml of 30% Trichloroacetic acid (TCA) was added followed by 0.5ml of 0.75% TBA. The mixture was then placed in a water bath at 80 0 C for 45 minutes and then cooled on ice before spinning at 3000 x g for 15 minutes. The absorbance of the reaction mixture was read in a spectrophotometer at 532 nm against distilled water which served as a reference blank. The amount of malondialdehyde molecules generated in the treated animals was determined with the molar extinction coefficient of 1.56 x 10 5 M -1 cm -1 , using the formula below: Absorbance olume of mixture nm olume of ample mg Protein

Superoxide dismutase test
The amount of superoxide dismutase present in the sample of liver cells of the animals administered with individual extract of A. indica, A. boonei and recipe was determined according to the previously described method (Mistra and Fridovich, 1972;Adekunle, 2012). Briefly, the homogenized sample of liver tissue was diluted in 10 fold dilutions, after which 0.2ml of the diluted sample was added to 2.5ml of 0.05M carbonate buffer (pH 10.2). Freshly prepared adrenaline (0.3ml) at 0.3mM was added to the mixture and was thoroughly mixed before equilibrated in the spectrophotometer. The reaction of the mixture was monitored for 150 seconds at 30 seconds intervals setting the absorbance at 480nm. The SOD activity was calculated as stated below: SOD Activity (Absorbance x olume of mixture) (Ɛ 480nm x Sample Vol x mg protein) = Unit/mg protein Molar extinction of OD at 48 nm (Ɛ 480nm ) = 525 M -1 cm -1

Catalase test
Catalase activity of A. indica, A. boonei extracts and the recipe was determined as previously described by Tadayuki et al. (2013). Four milliliters of 0.2 M H 2 O 2 solution (800 µmoles) was mixed with 5ml of 0.01 M Phosphate buffer (pH 7.0) in a flat bottom flask of 10 ml capacity. The reaction mixture was added with 1 ml of enzyme preparation, and subsequently with 2 ml of dichromate acetic acid at room temperature after every 1 minute.

Determination of total protein
Each of the test samples and calibrator (25 µl) was treated with 1 ml of Biuret solution, while the blank sample was 1 ml Biuret solution. After incubation of the mixtures at 37 o Cfor 5 minutes, the absorbance of the sample and calibrator were read against the blank using a spectrophotometer set at 540 nm (Caguioa et al., 2019). The total protein concentration was determined using the formula: Abs. of ample Abs. of tandard ( alibrator onc.) Where Abs = Absorbance Conversion factor (g/dL) X 144.9 Asian J. Med. Biol. Res. 2021, 7 (3) 252 2.6. Allium cepa assay Onions were purchased from wazo market, Ogbomoso. They were sun dried for two weeks to reduce moisture content so as to facilitate root growth. The onions were descaled carefully using a razor blade, leaving intact the primordial root ring (Akinboro et al., 2020b;Akinboro et al., 2021aAkinboro et al., , 2021b. The onions were rinsed with distilled water and wiped with tissue paper. Sixty onion bulbs were grown in distilled water inside 100 ml beakers placed inside a cupboard for 24 hours in order to initiate root growth (Akinboro et al., 2017). Thereafter, the onions were transferred into different doses (100%, 50%, 25% and 12.5%) of each of the plants extracts and the controls for another 24 hours root growth in a cupboard. The negative and positive controls were distilled water and sodium azide (0.05%), respectively. After 48 hours of root growth, roots from 4 onions were harvested and preserved in ethanol: glacial acetic acid (3:1) and stored at 4°C in a refrigerator for microscopic evaluation. The remaining six onions per dose were placed on fresh extracts, recipe and controls to continue their root growth for another 24 hours. After 72 hours of root growth, root lengths from the onions were cut from the base and measured with a meter rule. The percentage root growth relative to that of the negative control was calculated according to the formula below: root growth Average root length at a dose of an extract Average root length of negative control

Slide preparation
The fixed roots tips in ethanol: acetic acid (3:1) were rinsed in distilled water inside a petri dish to cleanse them of the fixative. They were then hydrolyzed inside 1N hydrochloric acid in a petri dish, then placed in an oven set at 6 ˚ for minutes. he hydrolyzed roots were treated for slides preparation as previously described (Akinboro et al., 2020b, Akinboro andJimoh, 2021a;Akinboro et al.,2021b). The prepared slides were observed under a binocular light microscope (PEC, Medical, USA) using immersion oil objective lens. A total number of 5,000 cells were counted and observed for stages of mitosis and chromosomal aberrations in each dose. Photomicrographs of the dividing stages and aberrations were taken with the help of a digital camera fixed on the microscope. The mitotic index of each treatment and control was calculated with the formula below:

FTIR analysis of antimalarial extracts and recipe
Each of the samples of A. indica, A. boonei, and recipe was transformed to Potassium bromide powdered tablets and then placed on mart R™ Attenuated otal Reflectance (A R) accessory at room temperature ( ° ). he prepared tablets were scanned using the Nicolet iS10 FTIR spectrophotometer (Thermo Fisher Scientific Inc, Madison, USA) coupled with deuterated triglycine sulfate (DTGS) detector and potassium bromide (KBr)/Germanium as abeam splitter. The spectra were scanned at wave-numbers of 4000-650cm-1 while connected to the software OMNICver.9.7. Data were recorded in three replicates using absorbance mode to facilitate quantitative analysis (Siregar et al., 2018).

Statistical analysis
The obtained data in terms of DNA fragmentation, mitotic index, chromosomal aberrations and root growth were summarized to mean, standard error and percentage, and then analyzed using ANOVA in the SPSS programme (version 17.0). Duncans multiple range comparison test was performed to determine the significant differences between the means of treatments and controls (p < 0.05).

Results
DNA fragmentation activity of the water extract of A. indica and the antimalarial recipe was inversely proportional to the doses, unlike the water extract of A. boonei which induced DNA fragmentations in ascending order of the doses (Figure 1). The percentage of fragmented DNA recorded at the selected doses except at 25.0% and 50.0% of the recipe was significantly different (p < 0.05) from that induced by the positive control. The water extract of A. indica, A. boonei and recipe significantly inhibited cell division in the roots of A. cepa as compared to the negative control ( Figure 2). However, this effect was not dose-dependent. Individual water extract of A. indica and A. boonei caused highest percentage mitotic index (MI) at 100.0% and 50.0%, respectively. The least percentage of MI induced by the recipe was obtained at 25.0%. Chromosomal aberrations (CA) such as anaphase bridge, sticky chromosomes and micronucleus were induced by the individual water extracts and their recipe. However, they were not dosedependent. The highest CA was induced at 12.5% and 50.0% doses of water extract of A.boonei and A. indica (Figure 3). Root growth of A. cepa was significantly inhibited by the water extract of A. indica, A. boonei and recipe at the selected doses except 12.5% recipe which induced a slightly higher but not significantly different (p > 0.05) root length from that recorded for the negative control. Water extract of A. boonei inhibited the root growth in this study better than extract of A. indica and the recipe (Figure 4). Super oxide dismutase activity of the water extract of A. indica, A. boonei and recipe was dose dependent and it was significantly different (p < 0.05) only at 100.0% dose of the individual extracts and recipe ( Figure 5). The effect of the individual extract of A. indica, A. boonei and recipe on induction of catalase was significantly higher (p < 0.05) at all the selected doses in this study than the controls ( Figure 6). The best catalase activity was recorded at100.0% recipe, however, this was not significantly different (p > 0.05) from the activity of water extract A. boonei at 50.0% and 100.0%. The ability of the extracts and recipe to generate malondialdehydes as products of lipid peroxidation of cell membrane showed that water extract of A. indica possessed the best inhibitory activity which was significantly different (p < 0.05) from that of A. boonei extract, recipe at the selected doses, and controls ( Figure 7).

Discussion
Safe consumption of herbal medicines by man is highly necessary, and it has to be established for their continuous ethnopharmacological uses. Plant extracts in herbal medicines are necessary to be screened for toxicity on cellular organelles and cell division individually and in combination so as to remove any of the recipe's constituents that has significant toxic effects rather than expected to be therapeutically effective. The individual water extract of A. indica, and A. boonei could be said to be non-mutagenic since the percentage DNA fragmentation induced by each of them was significantly lowered than that caused by distilled water (negative control), or had weak mutagenic activity having recorded significantly lowered percentage DNA fragmentation when compared to that induced by sodium azide (positive control). However, the recipe was more mutagenic than its two individual extracts as it induced DNA fragmentations that were singnificantly similar to that induced by sodium zide (positive control). Sodium azide is a mutagen capable of damaging DNA molecule when metabolized to an organic metabolite of azide called azidoalanine which may cause point mutation during DNA replication (Salim et al., 2009;Srivastava et al., 2011). The hydroxyl functional group detected in the extracts and the recipe suggests their richness in polyphenolic or phenolic phytochemicals which are known to possess good antioxidants properties. Extracts from leaves of A. indica and stem-bark of A. boonei were reported to contain phenolic and other kinds of phytochemicals (Akinmoladun et al., 2007;Awodele et al., 2010). The non mutagenic effect of the individual extracts of A. indica and A. boonei could be connected to the presence of antioxidants which are known to have good protective effect on DNA molecule thereby preventing the nucleic acid from undergoing mutation caused either through an inducing agent or spontaneously.In contrast,antioxidants may become pro-oxidants which readily cause DNA damage through free radicals generation in form of reactive oxygen species (Hussin et al., 2021). Free radicals are most often implicated in molecular damages inflicted on lipids, proteins and nucleic acids (Lin et al., 2013).The higher level of antioxidant activity of the recipe especially at 100% dose could be responsible for its induction of more DNA fragmentations than the individual extracts. Polyphenols and flavonoids act as antioxidants capable of scavenging free radicals andredox active metals ions chelation as well as indution of cellular antioxidant