In Vitro Anti-Venom Potentials of Aqueous Extract and Oils of Toona ciliata M. Roem against Cobra Venom and Chemical Constituents of Oils

There are high mortality and morbidity rates from poisonous snakebites globally. Many medicinal plants are locally used for snakebite treatment in Uganda. This study aimed to determine the in vitro anti-venom activities of aqueous extract and oils of Toona ciliata against Naja melanoleuca venom. A mixture of venom and extract was administered intramuscularly in rats. Anticoagulant, antiphospholipase A2 (PLA2) inhibition assay, and gel electrophoresis for anti-venom activities of oils were done. The chemical constituents of the oils of ciliata were identified using Gas chromatography-tandem mass spectroscopy (GC-MS/MS). The LD50 of the venom was 0.168 ± 0.21 µg/g. The venom and aqueous extract mixture (1.25 µg/g and 3.5 mg/g) did not cause any rat mortality, while the control with venom only (1.25 µg/g) caused death in 1 h. The aqueous extract of T. ciliata inhibited the anticoagulation activity of N. melanoleuca venom from 18.58 min. to 4.83 min and reduced the hemolytic halo diameter from 24 to 22 mm. SDS-PAGE gel electrophoresis showed that oils completely cleared venom proteins. GC-MS/MS analysis showed that the oils had sesquiterpene hydrocarbons (60%) in the volatile oil (VO) and oxygenated sesquiterpenes (48.89%) in the non-volatile oils (NVO). Some major compounds reported for the first time in T. ciliata NVOs were: Rutamarin (52.55%), β-Himachalol (9.53%), Girinimbine (6.68%) and Oprea1 (6.24%). Most compounds in the VO were reported for the first time in T. ciliata, including the major ones Santalene (8.55%) and Himachal-7-ol (6.69%). The result showed that aqueous extract and oils of T. ciliata have anti-venom/procoagulant activities and completely neutralized the venom. We recommend a study on isolation and testing the pure compounds against the same venom.


Introduction
The World Health Organization classified snakebite envenomation as one of the most neglected tropical diseases (NTD) in terms of incidence, severity, and clinical characteristics [1]. This has served as a basis for its advocacy and enlisting as an NTD under category A [1,2]. In Sub-Saharan Africa, Snakebite envenoming constitutes a serious medical condition that primarily affects residents of rural communities including Asia, Latin America, and New Probit Analysis to Determine LD 50 Probit analysis is a specialized form of regression analysis, which is applied to binomial response variables, i.e., variables with only one of two possible outcomes (positive/negative), that is, death/survival in this study.
Probit statistic was used to analyze the data in Table 1, and the result of the calculation is in Table 2. The result of Table 2 was plotted and graphed ( Figure 1). The regression equation was y = 3.3203x + 7.5693 which was used to calculate the LD 50 = 0.16833886 ± 0.210835 µg/g. Generally, the result of this study showed that N. melanoleuca venom is very lethal since it has LD50 as low as 0.16833886 µg/g.

Administration of a Mixture of Venom and Plant Extracts after the Pre-Incubation Test
Dose of control with a concentration of 1.25 mg/kg of venom killed rats with a mean Generally, the result of this study showed that N. melanoleuca venom is very lethal since it has LD 50 as low as 0.16833886 µg/g.

Administration of a Mixture of Venom and Plant Extracts after the Pre-Incubation Test
Dose of control with a concentration of 1.25 mg/kg of venom killed rats with a mean survival time of 2.0 h. The same concentration of venom (1.25 mg/kg) mixed with 3.5 mg/g of aqueous extract of T. ciliata, there was no death of rats registered. For the mixture of venom and a higher concentration of extract (5.0 mg/kg), the result showed no mortality after 48 h (Table 3), and the rats were taken to their cages to live normally under observation for more than 4 days. Experiments were performed in triplicate, and the MCD-P dose of 12.5 µg/mL gave a mean clotting time of 5.58 ± 0.44 min, but there were no clots formed for concentrations starting at 40 µg/mL and above. The control (Phosphate buffered saline, PBS) caused coagulation at 3.92 ± 0.38 min (Table 4). Generally, several proteins with enzymatic activity, such as PLA 2 and proteinases, inhibit blood coagulation, and this study showed that at high concentrations of N. melanoleuca venom ≥40 µg/g, there was inhibition of blood clots.
The effects of extracts on coagulation time revealed that concentrations of 25.5 and 100 mg/mL caused plasma coagulation without recalcification. Concentrations of 12.5, 6.25, 1, 0.5, 0.25, and 0.125 mg/mL did not cause coagulation. However, clots occurred in shorter periods than control samples when recalcified with 25 mM CaCl 2 (Table 5).
These results point out that higher doses cause coagulation without recalcification, which is unique, but the study required recalcification, which is why the low doses were chosen.

Inhibition of the Anticoagulation Activity of Venom
Neutralization of the anticoagulation activity of venom by the aqueous extract was compared with SAIMR (South African Institute for Medical Research) polyvenom (South African antisera) purchased from joint medical stores in Kampala, Uganda. MCD-P was multiplied by two (2xMCD-P) and constantly used without varying 2xMCD-P (25 µg/mL) caused coagulation for 11 min, while when 2xMCD-P mixed with 6.25, 0.5, and 0.125 mg/mL coagulation occurred at meant clotting time of 22.29, 14.65, and 14.83 and antisera at 8.67 min (Table 6). These indicate that the crude aqueous extract inhibited the coagulation activity of N. melanoleuca venom close to antisera as positive control. This is because the extract has proven to be a procoagulant (a precursor of a natural substance necessary for the coagulation of blood). The results of two experiments showed that a low concentration of T. ciliata extract has some activities, but due to using 2xMCD-P from other methods, we recommend using high doses of venom, which inhibits blood clots. In conclusion, some concentrations of aqueous plant extracts cause plasma clots, just as some low concentrations of venom. In the phospholipase A2 activity assay, N. melanoleuca venom was able to produce hemolytic haloes in agarose-human erythrocytes gels. The minimum hemolytic dose (MHD) is defined as the amount of venom that induces a hemolytic halo of 22 mm diameter [15]. N. melanoleuca venom had the lowest MHD of 2 µg and 10 µg at 10 mm and 22 mm diameter hemolytic halos, respectively. The MHD of N. melanoleuca venom was 10 µg. This shows that N. melanoleuca venoms have phospholipase A2 enzymes that can lyse human erythrocytes. The control 30 µL of PBS, pH 7.4, produced 6 mm halos (Table 7). Hemolytic properties of the aqueous extract of T. ciliata root bark on human erythrocytes showed that concentrations of ≤0.5 mg/mL produced no hemolytic halos, but concentrations of 1.0, 6.25, and 12.5 mg/mL produced hemolytic halos of 8, 16 and 18 mm (Table 8).  (Table 9) and Figure 2A,B.  Hemolytic activities of MHD were neutralized by an aqueous extract with a 2 mm diameter.
Extract at a concentration of ≥1.0 mg/mL causes small hemolytic activities but reduces the hemolytic activity of control (10 µg) from 24 to 22 halos.

Anti-Venom Activities of Non-Volatile and Volatile Oils of T Ciliata by SDS-PAGE
Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) (SDS-PAGE); SDS-PAGE was used to separate proteins with relative molecular masses from venom and protein band observed. Venom mixed with oils; lane three (V + VO) and lane four (V + NVO) there were no protein bands, while lane one Protein marker (PM) and lane Hemolytic activities of MHD were neutralized by an aqueous extract with a 2 mm diameter. Extract at a concentration of ≥1.0 mg/mL causes small hemolytic activities but reduces the hemolytic activity of control (10 µg) from 24 to 22 halos.

Anti-Venom Activities of Non-Volatile and Volatile Oils of T Ciliata by SDS-PAGE
Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) (SDS-PAGE); SDS-PAGE was used to separate proteins with relative molecular masses from venom and protein band observed. Venom mixed with oils; lane three (V + VO) and lane four (V + NVO) there were no protein bands, while lane one Protein marker (PM) and lane two venoms showed protein bands as control ( Figure 3).

Anti-Venom Activities of Non-Volatile and Volatile Oils of T Ciliata by SDS-P
Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (P PAGE); SDS-PAGE was used to separate proteins with relative molecular venom and protein band observed. Venom mixed with oils; lane three (V + four (V + NVO) there were no protein bands, while lane one Protein marker ( two venoms showed protein bands as control ( Figure 3). The good Protein marker (PageRuler™ Plus Prestained Protein Ladd kDa) had a good range of KiloDalton (KDa) from 10 to 250, and the equipm calibrated, but some of the venom proteins were not very visible, but the fr venom gave the bands hence more research because the Snakes were cage when the protein bands are not present in the gel wells after running a mixtu with another extract, it means the extract has neutralized the protein. The good Protein marker (PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa) had a good range of KiloDalton (KDa) from 10 to 250, and the equipment was well calibrated, but some of the venom proteins were not very visible, but the freshly milked venom gave the bands hence more research because the Snakes were caged. Generally, when the protein bands are not present in the gel wells after running a mixture of proteins with another extract, it means the extract has neutralized the protein.

Chemical Composition of Non-Volatile and Volatile Oils by GC-MS/MS
The compounds of the oils were identified by matching their spectra and retention indices (Kovats Index) with those of the authentic samples and literature value from the Wiley Library of Mass Spectra database of the GC-MS/MS system and published data.
During analysis of all the classes of compounds in NVOs and VO, the classes of chemical compounds were Sesquiterpene hydrocarbon, oxygenated sesquiterpene, furanocoumarins, and hydrocarbon alcohol, all of which are present in both NVO and VO (Table 14).

Discussion
Research to develop a treatment for local envenoming is a clinical priority and has focused on the application of natural products in Uganda. Herbalists have been using plants to make a decoction from either aerial, stems, or roots bark and administered orally in most cases [28,29]. The quantity of the decoction administered is not specific.

Discussion
Research to develop a treatment for local envenoming is a clinical priority and has focused on the application of natural products in Uganda. Herbalists have been using plants to make a decoction from either aerial, stems, or roots bark and administered orally in most cases [28,29]. The quantity of the decoction administered is not specific.

Phytochemical Screening
Most of the phytochemicals were detected both in the aqueous and organic extract of T. ciliata. Flavonoids and polyphenolic compounds detected in this study have the ability to bind to macromolecules, and some of these have shown the potential to inhibit PLA 2 s in other studies [30]. These include; quercetin, luteolin, kaempferol, isoquercitin and rutin [30]. Pure quercetin-3-O-rhamnoside from Euphorbia hirta has been found to have anti-venom activity [31].

In Vitro Anti-Venom Potentials of Aqueous Extract of T. ciliata
When aqueous extracts were mixed with a lethal dose of venom, it completely neutralized venom because i.m. administration had no mortality registered. The many compounds could have affected the structure proteins, non-proteins, and enzymes of N. melanoleuca, yet there was no effect of solvents. The most life-threatening pathology in elapid snakebite victims derives from neurotoxins, which can cause paralysis of the diaphragm and, eventually, asphyxiation [33]. There is an indication that some pure compounds may have anti-venom potentials if isolated and tested according to the reseach by Kankara [34].
Annona senegalensis Pers (family: Annonaceae) is used traditionally in Nigeria to treat victims of snakebite; it has been found that the extract mixed with venom inhibited (in vitro) the toxic signs induced by the cobra (Naja nigricotlis nigricotlis Wetch) venom [35]. The extract of the leaves of Guiera senegalensis was found to detoxify (in vitro) venom from two common northern Nigerian snake species, Echis carinatus and Naja nigricollis, and survival percentage was recorded within 24 h [36].

Inhibition of Anticoagulant Activities of Venom
Bites from elapid snakes typically result in neurotoxic symptoms in snakebite victims. Neurotoxins are, therefore, often the focus of research relating to understanding the pathogenesis of elapid bites. However, recent evidence suggests that some elapid snake venoms contain anticoagulant toxins, which may help neurotoxic components spread more rapidly [37]. African spitting Naja species significantly inhibited thrombin which has the ability to clot human fibrinogen and the impediment of Factor Xa's ability to clot recalcified plasma by forming a prothrombinase complex with Factor Va [38]. Factor Xa is the trypsin-like proteinase (serine protease enzyme) of coagulation that catalyzes prothrombin activation (prothrombin is a protein known as clotting (coagulation) factors (causes blood to clot) it is made by the liver while thrombin prevents blood clot. Factor Va is an essential protein cofactor of the enzyme factor Xa which activates prothrombin to thrombin during blood coagulation [39]. Venom effects on the coagulation cascade could cause anticoagulant effects, including inhibition of the blood coagulation cascade enzymes thrombin and Factor Xa [9]. Several cobra species have been shown to produce anticoagulant effects by inhibiting blood coagulation factors through the use of modified Group I phosopholipase A 2 (PLA 2 ) toxins [40]. For example, inhibitors of the enzymatic activities of Factor Xa and thrombin have been isolated from the non-spitting African cobra N. haje [41].
Many snake venoms comprise of different factors, which can either promote or inhibit the blood coagulation pathway. Coagulation disorders and hemorrhage belong to the most prominent features of bites of the many vipers; fractions of Iranian Echis carinatus venom delayed the prothrombine time and thus can be considered anticoagulant factors [42]. Some snake venoms contain toxins that are direct or indirect anticoagulants that inhibit the clotting process, thus increasing the risk of bleeding. However, other species with anticoagulant toxins coexist with coagulant and hemorrhagic toxins, thus producing a far less clear or diagnostic clinical laboratory picture [43].
In this study, it has been found that a high concentration of forest cobra (N. melanoleuca) venom from 40 µg/mL and above prevent blood plasma from clotting while dosing from 30 µg/mL and below cause blood plasma clot. The aqueous of T. ciliata was made to prolong the coagulant activity of 2xMCD-P (25 µg/mL).
Higher concentrations of the aqueous extract of T. ciliata cause blood plasma clots without recalcification, thus proving to be procoagulant (a precursor of a natural substance necessary to coagulate the blood). This could be useful for drug development. The procoagulant activity of T. ciliata root bark extract has proven its neutralization potentials that inhibit the anticoagulant activity of N. melanoleuca venom through its crude extract with groups of chemical compounds working synergistically. Concentrations (12.5-0.125 mg/mL) do not cause coagulation and are used for experiments with recalcification.

Inhibition of Phospholipase A 2 Activities
Phospholipase A 2 (PLA 2 ) is a ubiquitous enzyme that is an important component of snake venoms. These enzymes hydrolyze glycerophospholipids at the sn-2 position of the glycerol backbone, liberating lysophospholipids and fatty acids. Snake venom protein PLA 2 s displays a great variety of biological activities, including neurotoxic, myotoxic, antiplatelet, hemorrhagic, and anticoagulant effects [43].
The membranes of the erythrocytes can be affected by the consumption of bioactive compounds from herbs and medicinal plants [44]. In this study, the aqueous extract of T. ciliata root bark caused hemolysis of human erythrocytes at concentrations 6.25 and 12.5 mg/mL. This concentration reduced the minimum hemolytic dose (MDH) of 10 µg of PLA 2 by 8.33%, implying that the plant has an inhibitory effect on PLA 2 of the forest cobra of Uganda. This result reveals that hemolytic activity was inhibited by 2.0-and 1.0-mm halos in a concentration-dependent manner which implies that the aqueous extract has anti-phospholipase A 2 hemolytic activities, indicating that hemolysis was reduced by the plant. These results are in agreement with other similar research performed elsewhere. For instance, chloroform extract of Cyphostemma adenocoule through in vitro inhibited phospholipaseA 2 enzyme [45]. Galic acid (GA) and other tannins other isolated from Brazilian Anacardium humile have been shown to be effective inhibitors of snake venoms' toxic effects, and herein we demonstrated GA's ability to bind to and inhibit a snake venom PLA 2 , thus proposing a new mechanism of PLA 2 inhibition, and presenting more evidence of GA's potential as an anti-venom compound [46].

In Vitro Anti-Venom Potentials of Oils of T. ciliata by SDS-PADE
The interest of using SDS-PAGE was to determine the decreased intensity or disappearance of bands as well as the appearance of bands of different molecular weights in the lanes loaded with venom incubated with both oils for reliable indicators of the anti-venom activity or not. 1970. There were no protein bands seen in both lanes three and four. These indicated that there were no proteins, thus implying that both oils neutralized the N. melanoleuca venom proteins. It can be concluded that both oils have anti-venom potential. Volatile oils have a high yield during hydro-distillation and thus can be recommended for anti-N. melanoleuca venom, but more research should be performed on other species of Snake venom. Similar research was performed on essential oil and aqueous extract of Aloysia citriodora against Bothrops diporus venom. The SDS-PAGE results for the inhibition of proteolytic activity showed that essential oil was found to have much more active than plant extracts, but both neutralize the proteolytic activity [47]. Bothrops atrox venom proteins were investigated using SDS-PAGE electrophoresis if the proteins formed molecular complexes or were precipitated after being exposed to the Urospatha sagittifolia. The result showed that the protein band loses intensity as the dose of the extract is increased. This suggests both the precipitation of proteins and/or the formation of molecular complexes, which can be related to the inhibition of enzymatic activity. Not only SDS-PAGE analysis, Ethanolic extract of Urospatha sagittifolia (Araceae) reduces paw edema, skin hemorrhage and lethality induced by the crude venom on mice [48]. In this study, there was complete disappearance of the protein band, which concludes that the oils are potential anti-venom, more especially essential (volatile) oil, which is easily extracted from the root bark of T. ciliata.

Chemical Constituents of Non-Volatile and Volatile Oils of T. ciliata Root Bark by GC-MS/MS
The amount of oxygenated sesquiterpene is higher (48.89%) in non-volatile oils (NVOs) than in volatile oil (VO) (25%) due to the fact that they were extracted with n-hexane and concentrated using rotary evaporations and eluted through column chromatography. Volatile oils (VO) underwent only hydro-distillation, and Sesquiterpene hydrocarbon (60%) was the highest.
In NVOs, 83 compounds, out of which 15 major compounds selected by their percentage abundance, are reported for the first time in the genus Toona (Meliaceae) in this study about the root bark. However, according to literature, essential oils were previously identified in the leaves and stems of species Toona ciliata, Cedrela odorata, and Cedrela fissilis of the family Meliaceae [27]. The following compounds are the major ones in NVOs root bark with their medicinal values in relation to anti-venom; Rutamarin (52.55%) was found to be an effective inhibitor of human monoamine oxidase B (hMAO-B) with an inhibition percentage of 95.26% and also a study on molecular docking of (S)-rutamarin with hMAO-B showed that it binds stronger to the hMAO-B binding cavity [49]. Monoamine Oxidase B (MAOB) is involved in the breakdown of dopamine, a neurotransmitter implicated in reinforcing and motivating behaviors as well as movement. MAO-B inhibition is, therefore, associated with enhanced activity of dopamine, as well as with decreased production of hydrogen peroxide, a source of reactive oxygen species [50][51][52]. This, therefore, indicates that the major compound in NVOs has the ability to neutralize the neurotoxicity of venom, which inhibit the release of neurotransmitter from exocytosis of the synaptic vesicle at the presynaptic site or bind to the neurotransmitter receptor at the post-synaptic site. Another compound is Panasinsene (2.06%) which is the major sesquiterpene compound of Panax ginseng with medicinal and health benefits in preventing neurodegeneration. The beneficial effects of P. ginseng on neurodegenerative diseases have been attributed primarily to the antioxidative and immunomodulatory activities of its ginsenoside components [53,54].
In volatile oil (VO) from the root bark, a total of 49 compounds were identified. Generally, according to a literature search, there has been no research performed on the root bark of Toona species. Therefore, the result presented about VO reported for the first time about the chemical constituents of VO in the root bark of Toona species. The reason why this study focused on the anti-venom potentials of root bark came up as a result of Traditional Medicine Practitioners (TMPs) in Northern Uganda using it for the treatment of snakebites. Medicinal values of major compounds in VO include; Santalene (8.55%); Santalene sesquiterpene is identified for the first time in a large amount in C. lansium from Guangxi Province and revealing the presence of α-santalene in C. emarginata. The present work exhibited that Eos of Clausena has an excellent potential for application in the management of the booklice Liposcelis bostrychophila [7,55]. Most health benefits are for Santalol. Santalene, a major component of the sandalwood essential oil, is a typical representative of sesquiterpenes and has important applications in medicine, food, flavors, and other fields, and it has been biosynthesized because of limited supplies. B-Copaene (8.1%); for the first time, a study reports that copaene is not genotoxic and it increases the antioxidant capacity in human lymphocyte cultures in a study conducted on the cytotoxic, genotoxic/antigenotoxic, and antioxidant/oxidant activity of copaene [56]. In this study, volatile essential oil neutralized cobra venom (SDS-PAGE analysis), and other essential oils also did neutralize the venom of other species. Essential oils have anti-venom potentials, essential oils from C. bonariensis and T. diversifolia inhibited the coagulant activity of B. atrox venom by increasing the clotting time from 100.8 to 264.0 s, respectively, and the oils from Ambrosia polystachya and Baccharis dracunculifolia caused 100% of inhibition on the fibrinogenolysis induced by Bothrops moojeni and Lachesis muta venoms [57]. Essential oils of Artemisia herba-alba and A. campestris (Asteraceae) inhibited inflammation and cytotoxicity induced by Cerastes cerastes venoms [58].

Ethical Approval
Ethical approval for this study was obtained from Gulu University Research Ethics Committee (GUREC) (No. GUREC-003-20) and the Uganda National Council for Science and Technology (UNCST) (No. SS 5207) for the use of animals and human erythrocytes models. The use of poisonous snakes for milking venom was obtained from the Uganda Wildlife Authority (UWA) (No. COD/96/02).

Milking and Lyophilizing of Venom
Healthy poisonous forest cobras (Naja melanoleuca) in the Elaphidae family were identified, selected, and quarantined by a herpetologist from UWA. The selected snakes were kept caged in captivity at Kavumba Wildlife Conservation and Research Centre in Wakiso district, central Uganda. All the standard safety precautions, including protective gear and antisera, carried in case of accidental snakebites, were taken prior to, during, and after handling the snakes. Milking of venom was performed following the method of Theakston and Reid [15] by pressing venom glands but with some modifications. The volume of venom milked per bite was measured and put in labeled sample bottles and kept in cold chain at 4-5 • C. The samples were then freshly transported to the Chemistry Department at Makerere University. The venom obtained was lyophilized to dryness were kept in a refrigerator at 4-8 • C.

Source of Plants Material
The selection of plant material was based on a previous ethnopharmacological survey which showed T. ciliata to be one of the most frequently used plants for the treatment of all kinds of poisonous snakebites in Uganda [28]. A voucher specimen of the plant was collected using the WHO standard plant collection guidelines on good agricultural and collection practices for medicinal plants [61] and deposited at the Makerere University herbarium for identification (Voucher No. 50912/ODF 012). After identification, Toona M.Roem was also checked with http://www.theplantlist.org/tpl1.1/search?q=Toona+ ciliata (accessed on 1 March 2023). Collection and of data was from 5 August to 15 October 2017. The roots of T. ciliata were harvested from Namokora sub-county in Kitgum district, northern Uganda, following the same WHO guidelines. The root barks were cleaned and shade dried for 2 weeks. The dried sample was pulverized into fine powder and then kept for extractions.

Extraction of the Plant Material
Extraction of 100 g of powdered root bark of T. ciliata was performed twice using warm distilled water (soaked and left for 12 h). The extract was filtered using filter funnel packed with cotton wool then filtrate was filtered using Whatman No. 1 filter paper. The filtrate of the aqueous extracts was freeze-dried and kept in fridge (−8 • C) for phytochemical screening and anti-venom potential analysis. Organic extraction was performed with a solvent system made of Dichloromethane (DCM) and ethylacetate (EA) in a ratio of 1:1 and used for the extraction of 0.5 kg of dry powdered root bark of T. ciliata. Filtration was performed using Whatman No. 1 filter paper, and the filtrate was concentrated at reduced pressure at 30-40 • C with a rotary evaporator model RE100-Pro China [62]. The dry sample was kept for phytochemical screening and isolation of fractions [63].

Phytochemicals Screening
The aqueous and organic extracts of root bark of T. ciliata were screened for alkaloids, flavonoids, tannins, flavonoids, anthraquinones, coumarins, and terpenoids using standard methods [64]. In order to find out whether the plants extract neutralized venom, an experiment was run using venom concentration of 1.25 µg/g as control. The solvent, normal saline, used as a control in LD 50 determination showed no mortality, and it was left out. The stock solution of venom in normal saline was made at a concentration of 1 mg/mL, and that of plant extract was made at 100 mg/mL. A concentration of 1.25 µg/g (from 1 mg/mL) venom dose was mixed with 3.5 mg/g (100 mg/mL) aqueous extract. Similarly, A concentration of 1.25 µg/g (from 1 mg/mL) venom was mixed with 3.5 mg/g (100 mg/mL) aqueous extract. The mixtures of venom and aqueous extract were incubated at 25 • C for 20 min and centrifuged (Centrifuge 5424R, Germany) at 2000 rpm for 10 min. Group I was administered with control venom only. Group II was administered with the resultant mixture intramuscularly and observed for 48 h and days [65,66]. The minimum coagulation dose of plasma (MCD-P) of the venom was determined by first evaluating the coagulation activity of T. ciliata extracts as described by the method of Theakston and Reid [15]. Human plasma collected from Nakasero blood bank in Kampala, Uganda, was citrated by adding 4% trisodium citrate as an anticoagulant. The lyophilized venom was used to make a stock solution of 1 mg/mL from which different concentrations from 1-120 µg/mL were diluted with phosphate-buffered saline (PBS) at pH 7.4. Various concentrations of 12.5, 25, 30, 40, 50, and 100 µg/mL of the venom were mixed with 200 µL of the plasma and incubated at 37 • C for 30 min. The plasma was re-calcified to remove anticoagulants with 25 mM CaCl 2 . The PBS alone was used as a control. The setup was observed every 15 s by gentle tilting until a solid thrombus formed, and this was recorded as a recalcification time in minutes. The minimum amount of venom that clots or that allows the formation of a complete thrombus was taken as the minimum coagulation dose of plasma. MCD was calculated by plotting clotting time against venom concentration and reading the level at the 60 s clotting time [15,66].

Determination of Effects of T. ciliata Extract on Coagulation of Plasma
To determine if T. ciliata extracts have effects on the clotting times of plasma, 100 µL of extracts of concentrations 100, 50, 25, 12.5, 6.25, 1, 0.5, and 0.25 mg/mL were mixed with 200 µL of citrated plasma and incubated in a water-bath at 37 • C. The setup was observed every 5 min for the first 1 h and every 10 min for 2 more h. Coagulability and incoagulability were recorded. After 3 h of observation, 100 µL of 25 mM CaCl 2 was added to the unclotted plasma samples, and the recalcification time was determined to evaluate the effect on coagulation time.

Neutralization of Anticoagulation Activity of Venom
The minimum coagulation dose of venom determined was doubled (2 MCD-P) and mixed with varied concentrations of plant extracts (6.25, 1, 0.5, 0.25, and 0.125 mg/mL) incubated for 30 min at 37 • C. Then 100 µL of the resultant mixture was added to 200 µL of citrated plasma and re-calcified by the addition of 100 µL of 25 mM CaCl 2 . The clotting times were recorded by gently tilting the test tube containing the sample every 15 s till coagulation took place [15,66]. Evaluation of phospholipase A2 activity of venom by indirect hemolytic activity was assayed as described by Theakston and Reid [15]. A human blood agar gel plate was made containing (300 µL Egg yolk, 0.01 M CaCl 2 and 1% (w/v) of Nutrient agar) as follows; 300 µL egg yolk (6 egg yolk/l) solution was added to the normal saline solution, then mixed with 250 µL of 0.01 M CaCl 2 solution was in a ratio of 1:3 then added to 25 mL of 1% (w/v) of Nutrient agar (powder) at 50 • C dissolved in PBS. Packed human erythrocytes (300 µL) were washed four times with normal saline solution and then added to the resultant mixture of egg yolk, CaCl 2 and agar. The mixture was poured into a petri dish and allowed to form a gel (agarose-egg yolk-human erythrocyte gels plate). Then, 6 mm diameter wells were made and filled with 30 µL of venom samples. After 20 h of incubation at 37 • C, the diameters of hemolytic halos were measured. In order to determine the MHD of venom, 30 µL of solutions containing different amounts of venom concentrations (2,10,12,14,16,18, and 20 µg/mL) was applied into the wells. Control wells contained 30 µL of PBS, pH 7.4. After 20 h of incubation at 37 • C, the diameters of hemolytic halos were measured. The MHD was determined as the amount of venom that induced a hemolytic halo of 22 mm diameter [20]. The determined MHD was used with different concentrations of the extracts alone to determine the hemolytic properties of T. ciliate extract.

Neutralization of Minimum Hemolytic Dose of Venom
The constant MHD of venom was mixed with varied concentrations (6.25, 1.0, 0.5, 0.25 mg/mL) of the aqueous extracts of T. ciliate and incubated for 30 min at 37 • C. The aliquots of the mixture, about 15 µL, were added to wells of agarose-egg yolk-human erythrocyte gels plate and then incubated for 20 h at 37 • C. The MHD of the venom only was used as the control. Venom neutralization was calculated as the percentage inhibition that reduces 50% of the diameter of the hemolytic halo compared to that of the control [15,66].
The Thin layer chromatography (TLC) Allugram XTra-sheets SIL G/UV254 (20 × 20 cm) to check the homogeneity of fractions. Fractions were combined, concentrated with a digital rotary evaporator, and dried. Three non-volatile oil was isolated with a solvent system of Hex: EA = 4:1 and 3:2.

Hydro-Distillation of Volatile Oils from Root Barks of T. ciliata
Air-dried and pulverized root barks (88.8 g) of T. ciliata was hydro distilled using a Clevenger-type glass apparatus for 3 h by following the British Pharmacopoeia [67,68]. The distilled oil was 3.0 mL (3.10%) (weight or density in drop (s) not performed, but the density of oils is 0.918 mL/g) obtained were packed in amber bottles and kept in a refrigerator until analysis.

Anti-Venom Activities of Non-Volatile and Volatile Oils of T. ciliata by SDS-PAGE SDS-PAGE Gel Electrophoresis
Samples were prepared as Lyophilized venom was dissolved in normal saline at a concentration of 1 mg/mL; both non-volatile and volatile oils were dissolved in DMSO at a concentration of 20 µL/mL. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) (SDS-PAGE) was carried out using a Mini-Protean IV Electrophoresis Cell device under denaturing conditions using 12% (w/v) stacking gel solution (8% acrylamide and 0.8% N,N'-bis-methylene acrylamide) and 4% (w/v) separating gel solution 0.37 M Tris-HCl (pH 8.8) and 0.1% SDS) [68]. The venom and both oils were mixed in a ratio of 1:2 (venom: oils) and incubated for 20 min at room temperature. A protein loading buffer (Protein marker) was added to each of the samples in the tubes and then heated for ten min. The samples were loaded on SDS PAGE gel as follows; Protein marker, Venom only as control, Venom Mixed with NVO, Venom mixed with VO and samples run for 75 min at a constant voltage of 150 v. The gels were then stained with 0.25% commassie brilliant blue overnight. The gels were then destined for 1-and 30-min using the method of Laemmli [68]. Decreased intensity or disappearance of bands as well as the appearance of bands of different molecular weights in the lanes loaded with venom and extracts/oils were used as reliable indicators of activity. The non-volatile and volatile oils soluble in n-hexane analytical grade were autoinjected to Gas chromatography-tandem mass spectroscopy (GC-MS/MS) for analysis.
Analysis of the oils was performed using GC-MS/MS (model GCMS-TQ 8040, Shimadzu, Japan equipped with autosampler coupled with detectors MS/MS. Capillary column was a DB-5 MS UI fused silica capillary of length 30 m, diameter 0.25 mm, and thickness 0.25 µm with 5% phenylmethylsiloxane stationary phase. Injection method: injection mode was splitless, sampling time was 2.0 min. Pressure set at 120.0 kpa, and column flow was 1.77 mL/min. Helium was used as the carrier gas at a flow rate of 1 mL/min. The split ratio was 1:0. One microliter of the diluted oils (in hexane) was injected for analysis. n-Alkane of C8 to C30 were run under the same condition of Kovats indices determination as control, the total GC-MS/MS program time was 35.0 min.
Program temperature: The initial oven temperature of the column was set at 60 • C and was heated to the final temperature of 315 • C at a rate of 5 • C/min. The injection temperature was 250 • C. Mass spectroscopy parameter: Ion source temperature was 230 • C, interface temperature was 250 • C, solvent cut time 3 min., scan speed was 1666 at Acq. Mode og Q 3 scan. Scan time was 60 min with a scanning range of 35-450 amu.

Identification of Chemical Compounds of Both Non-Volatile and Volatile Oils
The constituents of both NVO and VO were identified by GC using retention indices compared with those of the literature. The retention indices were determined in relation to a homologous series of alkanes under the same operating conditions. The components of the oils were identified by matching their spectra and retention indices (Kovats Index) with those of the authentic samples and literature value from the Wiley Library of Mass Spectra database of the GC/MS system and published data [69].

Conclusions
The compounds in aqueous extract and oils neutralized forest cobra (N. melanoleuca) venom. This was evidenced by the aqueous extract of T. ciliata neutralizing venom after Pre-incubation of venom with aqueous extract. Since aqueous extract causes blood cloth at some doses, it can be used as a procoagulant drug (a precursor of a natural substance necessary for the coagulation of blood), and it has the anticoagulation activity of venom close to antisera (standard drug). The volatile and non-volatile oils have anti-venom potential; therefore, they can be a good topical application as anti-venom for forest cobra bites. Generally, there has been no research performed on the root bark of Toona species. Therefore, aqueous extract and oils of T. ciliata showed high potential to be developed as anti-venom and aqueous extract as a coagulant herbal drug. Aroma is one of the most important attributes of food pharmaceuticals and is directly associated with product acceptance by consumers. T ciliata has a good yield of essential oil (3.10%) from the root bark. Therefore, this study provides research data for the application of aroma to pharmaceutical products of anti-venom drugs. We recommend future researchers make herbal formulations of medicine with this plant extract for anti-venom and procoagulant. The limitations of this study were the difficulty of hunting wild cobra, which was why we used cage ones, and SDS-PAGE was not showing all the proteins of forest cobra (N. melanoleuca) venom clearly.  Data Availability Statement: Supporting data to this article is publicly available in the Mendeley data repository [70]: Data, V 1, doi:10.17632/n9f4cj8mrn.1.