Exploring nature’s antidote: unveiling the inhibitory potential of selected medicinal plants from Kisumu, Kenya against venom from some snakes of medical significance in sub-Saharan Africa

Background: The present study investigated the efficacy of Conyza bonariensis, Commiphora africana, Senna obtusifolia, Warburgia ugandensis, Vernonia glabra, and Zanthoxylum usambarense against Bitis arietans venom (BAV), Naja ashei venom (NAV), and Naja subfulva venom (NSV). Methods: 40 extracts and fractions were prepared using n-hexane, dichloromethane, ethyl acetate, and methanol. In vitro efficacy against snake venom phospholipase A2 (svPLA2) was determined in 96-well microtiter and agarose-egg yolk coagulation assays. in vivo efficacy against venom-induced cytotoxicity was determined using Artemia salina. Two commercial antivenoms were used for comparison. Results: The 96-well microtiter assay revealed poor svPLA2 inhibition of BAV by antivenom (range: 20.76% ± 13.29% to 51.29% ± 3.26%) but strong inhibition (>90%) by dichloromethane and hexane fractions of C. africana, hexane and ethyl acetate extracts and fraction of W. ugandensis, dichloromethane fraction of V. glabra, and the methanol extract of S. obtusifolia. The methanol extract and fraction of C. africana, and the hexane extract of Z. usambarense strongly inhibited (>90%) svPLA2 activity in NAV. The hexane and ethyl acetate fractions of V. glabra and the dichloromethane, ethyl acetate, and methanol extracts of C. africana strongly inhibited (>90%) svPLA2 in NSV. The agarose egg yolk coagulation assay showed significant inhibition of BAV by the dichloromethane fraction of C. africana (EC50 = 3.51 ± 2.58 μg/mL), significant inhibition of NAV by the methanol fraction of C. africana (EC50 = 7.35 ± 1.800 μg/mL), and significant inhibition of NSV by the hexane extract of V. glabra (EC50 = 7.94 ± 1.50 μg/mL). All antivenoms were non-cytotoxic in A. salina but the methanol extract of C. africana and the hexane extracts of V. glabra and Z. usambarense were cytotoxic. The dichloromethane fraction of C. africana significantly neutralized BAV-induced cytotoxicity, the methanol fraction and extract of C. africana neutralized NAV-induced cytotoxicity, while the ethyl acetate extract of V. glabra significantly neutralized NSV-induced cytotoxicity. Glycosides, flavonoids, phenolics, and tannins were identified in the non-cytotoxic extracts/fractions. Conclusion: These findings validate the local use of C. africana and V. glabra in snakebite but not C. bonariensis, S. obtusifolia, W. ugandensis, and Z. usambarense. Further work is needed to isolate pure compounds from the effective plants and identify their mechanisms of action.


Introduction
An estimated 5 million people are bitten by snakes every year, about half of whom experience clinical illness, and up to 140,000 die from complications related to envenomation (Chippaux, 1998;Kasturiratne et al., 2008).Snakebites are prevalent among lowincome individuals residing in rural, tropical areas with limited access to healthcare (Oliveira et al., 2023).Consequently, local people frequently rely on folk medicine, which includes the use of medicinal plants.Several such plants, including C. bonariensis, C. africana, S. obtusifolia, Warburgia ugandensis, Vernonia glabra, and Zanthoxylum usambarense have gained notoriety among the Luo people in Kisumu, Kenya, due to their putative anti-snake venom properties (Owuor et al., 2005;Owuor and Kisangau, 2006).These plants are known by the locals as "yadh asere" (C.bonariensis), "arupiny" (C.africana), "olusia" (V.glabra), "sogo" (W.ugandensis), and "roko" (Z.usambarense).They have widespread ethnomedicinal use locally including in snakebite and share phylogenetic relationships with plants previously reported as antisnake bite remedies, e.g., Senna siamea, Conyza sumatrensis, and Zanthoxylum chalybeum (Owuor and Kisangau, 2006).Treatments include the use of cut, suck, and bind techniques, followed by the application of plant leaf and root poultices secured with bark or cloth strips (Owuor et al., 2005).However, there is a general concern about the efficacy and safety of alternative remedies in managing diseases (Puzari et al., 2022).Rigorous scientific scrutiny of these remedies is essential to determine the validity of the ethnomedicinal claims and to ensure the development of safe and efficacious interventions for snakebite victims (Puzari et al., 2022).
B. arietans, N. ashei, and N. subfulva are snakes of medical importance in sub-Saharan Africa (Calvete et al., 2007;Currier, 2012;Tasoulis and Isbister, 2017;Onyango, 2018;Okumu et al., 2020;Dyba et al., 2021) (Figure 1).Antivenom is the mainstay of treatment for envenomation by these snakes but is expensive, has limited availability, and does not sufficiently neutralize some key venom toxins, e.g., cytotoxins which cause dermonecrosis in snake bite victims.Medicinal plants are used to plug this gap, but they lack scientific validity.This study employed a combination of in vitro and in vivo methods to evaluate the antivenom properties of C. bonariensis, C. africana, S. obtusifolia, W. ugandensis, V. glabra, and Z. usambarense against B. arietans, N. ashei, and N. subfulva venoms.

Collection and identification of medicinal plants
Plant materials were collected in November 2016 in Kisumu County.The East African Herbarium in Nairobi, Kenya identified and verified the plant specimens, as shown in (Supplementary Figure S1) (Supplementary Section).REF NMK/BOT/CTX/1/2/1.The selection of the plants was based on five factors: 1) their extensive local ethnopharmacological use in treating snakebites; 2) their evolutionary link to other plants used for the same purpose; 3) the findings of an Owuor and Kisangau survey on the use and practice of herbal medicine (Owuor and Kisangau, 2006), 4) the lack of published research outlining the plants' bioactive ingredients, and 5) their availability for evaluation.An overview of the plants used in this study is as shown in (Figure 2).

Preparation of plant material
After being cleaned to get rid of any dust that stuck to them, the plant materials were shade-dried and then ground into a powder using an electric mill (Retsch Grindomax, Germany).

Soxhlet extraction of the medicinal plants
Powdered plant materials were sequentially extracted by Soxhlet extraction using n-hexane, dichloromethane, ethyl acetate, and methanol and concentrated under reduced pressure at 40 °C on a rotary evaporator (Stuart, Cole-Parmer-UK) (Janardhan et al., 2014).The percentage yield of the extracts was calculated as %w/w.

Extraction of medicinal plants using a modified maceration technique
Powdered plant materials were separately mixed with methanol, macerated for 72 h, and concentrated at 40 °C under reduced pressure on a rotary evaporator (Stuart, Cole-Parmer-  Frontiers in Pharmacology frontiersin.org04 UK).The methanol extracts were separated into four parts, distributed in de-ionized water, partitioned sequentially with n-hexane, dichloromethane, ethyl acetate, and concentrated under reduced pressure at 40 °C on a rotary evaporator (Stuart, Cole-Parmer-UK) (Alsayari et al., 2018).The percentage yield of the extracts was calculated as %w/w.

Ethics
The biosafety, animal care, and use committee of the University of Nairobi was consulted before the authors handled any experimental animal, as shown in Supplementary Figure S2 (Supplementary section) (REF BAUEC/2019/220).

Snake venom
Nine specimens of the large brown spitting cobras (N.ashei), Eastern Forest cobras (N.subfulva) and puff adders (B.arietans) were collected in the wild and identified by a herpetologist at Bioken snake farm, Kenya.Venom was collected from these snakes using the beaker method, snap frozen, lyophilized (Labconco, USA), and kept as a powder at −20 °C until it was reconstituted in phosphate buffered saline.

Determination of the in vitro anti-snake venom phospholipase A 2 activity of the prepared extracts
The 96-well microtiter plate assay The methods of Iwanaga and Suzuki (Iwanaga and Suzuki, 1979) and Molander and colleagues (Molander et al., 2014) were used.10 μL of a 10 μg/mL concentration of each of the venoms (in 0.1 M phosphate buffered saline) and 20 µL of a 100 μg/mL concentration of each of the prepared extracts were micro pipetted (Finnpipette, Thermo Fisher Scientific, USA) into 96-well microtiter plates (Costar ® 3590, USA) before 200 µL of a 1.1% egg yolk suspension in 0.1 M PBS adjusted to pH 8.1 and 0.2 mM CaCl 2 was added to each well, and the absorbance of the mixtures was taken at 620 nm on a multi plate reader (Thermo Fisher Multiskan, USA).The plates were incubated (Memmert, Germany) at 37 °C for 20 min and the absorbance measured again at 620 nm.svPLA 2 activity was measured as the decrease in turbidity of the egg yolk suspension from 0 to 20 min.The inhibition of svPLA 2 activity by the extract was expressed as percentage inhibition of enzymatic activity taking the absorbance of a well to which no venom was added as 100%.Extracts were tested in triplicate and antivenom was used as a positive reference.

The agarose-egg yolk coagulation assay
Extracts with >90% inhibition of the svPLA 2 activity in the aforementioned assay were further evaluated in the agarose egg yolk coagulation assay described by Habermann and Hardt (Habermann and Hardt, 1972) as follows.
These mixtures were micro pipetted into 0.5 mm wells on an agarose-egg yolk medium and incubated (Memmert, Germany) at 50 °C for 24 h.10% Carbol Fuchsin was used to visualize the enzymatic halos in each group and the diameter of the enzymatic halos was measured using a digital vernier calliper (Rolson, United Kingdom) and expressed as the minimum phospholipase concentration (MPC) i.e., the least dose of venom which is responsible for an enzymatic halo of 10 mm in the case of BAV and 15 mm in the case of NAV and NSV.

Cytotoxicity of the venoms, extracts, and antivenoms in Artemia salina
The in vivo toxicities of the extracts, venoms, and antivenoms were evaluated in Artemia salina according to the method described by Meyer et al. (1982) with modifications as described by Nguta et al. (2014).This was replicated in 5 different sample tubes for each venom, extract, or antivenom concentration.Physiological buffer saline (1 mL) was used as the negative control and vincristine sulphate was used as the positive control.

Neutralization of Artemia salina venominduced cytotoxicity by the extracts and antivenom
The WHO pre-incubation neutralization protocol was used and adjusted to A. salina (WHO, 2016).Varying doses of the extracts or antivenom (50 μg/mL, 100 μg/mL, 200 μg/mL, 400 μg/mL, and 800 μg/mL) were incubated (Memmert, Germany) with a 2LC 50 dose of each of the venoms at 37 °C for 30 min.The resulting mixtures were added to vials containing A. salina and the survivors were counted after 24, 48, and 72 h of exposure.The median effective concentration of the extracts was defined as the minimum amount of extract (in µL) required to neutralize 1 mg of venom.

Quantitative phytochemical composition
Total phenolics, flavonoids, glycosides, and tannins were estimated using a UV-VIS spectrophotometer (Spectronic 21-D, USA).Analytical grade gallic acid, catechin, and rutin were used as standards.

Determination of total phenolic content (TPC)
The method of Harnafi et al. was used (Harnafi et al., 2008).The extracts/fractions were mixed with 7.5% w/v Na 2 CO 3 solution and 2.5 mL of Folin-Ciocalteau reagent (FINAR, India), and the absorbance was read at 765 nm on a UV-VIS spectrophotometer (Spectronic 21-D, USA) and a gallic acid standard curve was generated.The assay was performed in triplicate and the results were expressed as milligrams of Gallic acid equivalents per Gram of the dry plant material (mg.GAE.g -1 ).

Determination of total flavonoid content (TFC)
The method of Atanassova et al. was used (Atanassova et al., 2011).The extract/fractions were mixed with distilled water, 5% w/v sodium nitrite (NaNO 2 ), 10% w/v aluminum chloride (AlCl 3 ), and 1 M sodium hydroxide (NaOH), and the absorbance was read on a UV-VIS spectrophotometer (Spectronic 21-D, USA) at 510 nm.The flavonoid content was determined from a catechin standard curve.The assay was performed in triplicate and the results were calculated as milligrams of Catechin equivalents per Gram of the dry plant material (mg.CE. g -1 ).

Tannin content
The method of Amadi et al. was used (Amadi et al., 2004).The extracts/fractions were boiled gently for 1 h and mixed with 2.5 mL of Folin-Denis reagent, 5 mL of saturated Na 2 CO 3 solution, and 25 mL of distilled water.The mixture was left to stand for 30 min in a water bath (Memmert, Germany) at 25 °C and the absorbance was read on a UV-VIS spectrophotometer (Spectronic 21-D, USA) at 700 nm.The tannin content was determined from a tannic acid standard curve.The assay was performed in triplicate and the results were calculated as below: Tannic acid mg 100g C × extract volume × 100 Aliquot volume × weight of sample Where C is concentration of tannic acid read off the graph.

Cardiac glycoside content
The method described by Muhamad and Abubakar (Muhammad and Abubakar, 2016) was used.The extracts/ fractions were mixed with distilled water, 12.5% lead acetate, 47% w/v Na 2 HPO 4 , and Baljet reagent (95 mL of 1% picric acid+5 mL of 10% NaOH).A blank titration was carried out using 10 mL distilled water and 10 mL Baljet reagent (95 mL of 1% picric acid+5 mL of 10% NaOH).This mixture was allowed to stand for 1 hour and the absorbance was read on a UV-VIS spectrophotometer (Spectronic 21-D, USA) at 495 nm.The percentage (%) of total glycosides present in extracts/fractions was calculated as % of total glycosides= (A×100)/77 g %.Where A = absorbance of samples.

Data analysis
The effect of each of the extracts/fractions/antivenoms on the minimum phospholipase concentration of venom (s) was compared using one way-ANOVA and Dunnet's multiple comparison test.The lethality of venoms, extracts, fractions, and antivenoms in A. salina and their capacity to neutralize venom-induced cytotoxicity in the same model was analyzed using probit regression analysis.
Results on the phytochemical composition of the extracts/fractions were summarized in a table.p < 0.05 was considered significant.

Results
The percentage yield of extracts The percentage yield of the hexane root extract of C. africana prepared by the Soxhlet method was the lowest (0.23%), while the percentage yield of the dichloromethane leaf extract of V. glabra prepared by the maceration method was the highest (54.65%), as observed in (Supplementary Table S1).

Information on the snakes whose venom was used in the study
Most of the snakes used in this study were sourced from the Watamu area in Kenya.(Supplementary Table S2) in vitro microtiter well svPLA 2 neutralization assay.
>90% anti-svPLA 2 inhibition was observed against NAV with the methanol extract and fraction of C. africana stem bark, the methanol extract from the C. africana bark, and the hexane extract of Z. usambarense leaves.
>90% anti-svPLA 2 inhibition was noted against NSV with hexane and ethyl acetate fractions of V. glabra leaves and dichloromethane, ethyl acetate, and methanol extracts of C. africana bark (Table 1).

Cytotoxicity of the extracts, fractions, and antivenom in Artemia salina
The methanol extract of C. africana stem bark, the hexane extracts of V. glabra leaves and Z. usambarense leaf stalk were cytotoxic to A. salina with LC 50 values of 611.72 (251.06-3437.50)

Qualitative phytochemical composition of extracts and fractions
Flavonoids, phenolics, glycosides, and tannins were found to be present in the dichloromethane and methanol fractions of C. africana stem bark, the methanol extract of C. africana bark, and the ethyl acetate extract of V. glabra leaves.However, alkaloids, carboxylic acids, phytosterols, and terpenoids were absent in the extracts and fractions (Table 3).

Quantitative phytochemical composition of the non-cytotoxic extracts and fractions
The ethyl acetate extract of V. glabra leaves had the highest glycoside (0.003%), total flavonoid (2.990 mg/g catechin equivalents), and tannic acid content (0.010%) while the methanol extract of C. africana stem bark had the highest phenolic content (2.180 mg/g gallic acid equivalents) (Table 4).

Neutralization of venom-induced cytotoxicity by extracts, fractions, and antivenom
The dichloromethane fraction of C. africana stem bark had an effective concentration of 336.12 ± 59.97 μg/mL against BAVinduced cytotoxicity in A. salina.The methanol extract of C. africana bark was the most effective against NAV-induced cytotoxicity in A. salina with an EC 50 of 221.37 ± 30.33 μg/mL.The ethyl acetate extract of V. glabra leaves had an effective concentration of 329.39 ± 15.92 against NSV-induced cytotoxicity in A. salina.However, the test antivenoms were ineffective in neutralizing BAV, NAV and NSV-induced cytotoxicity in A. salina (Table 5).
The A. salina model has been used to evaluate the cytotoxicity of medicinal plants (Nguta et al., 2011;Mwangi et al., 2015), environmental contaminants (Barahona and Sanchez-Fortun, 1999;Sanchez-Fortun andBarahona, 2009), andvenom (Damotharan et al., 2015;Okumu et al., 2021).The present work was a continuation of our previous work where we investigated the capacity of two antivenoms to neutralize NAV-induced cytotoxicity in A. salina (Okumu et al., 2020).Moreover, we showed in another study that the A. salina model was a good surrogate for dermonecrosis in mice (Okumu et al., 2021).The present study established that some extracts and fractions of C. africana were effective in prolonging the survival of A. salina exposed to NAV.Isa and colleagues in a previous research reported that the crude methanol extract and fraction of C. africana dose-dependently neutralized N. nigricollis envenomation in mice (Isa et al., 2022).Abdullahi et al. reported the anti-snake venom properties of a C. africana related plant, i.e., Commiphora pedunculata against N. nigricollis venom (Abdullahi et al., 2017).While this study has highlighted the capacity of the prepared extracts to neutralize key effects of medically important sub-Saharan snakes, it did not evaluate the capacity of the extracts/fractions to neutralize other key toxins in the studied snake venoms including protease, hyaluronidase, and neurotoxins (3FTx's).Moreover, further work is needed to understand the identity of the compounds responsible for the observed extract/fraction induced cytotoxicity in A. salina.

Conclusion
These findings validate the local use of C. africana and V. glabra in snakebite envenomation and provide a basis for further work aimed at isolating pure compounds from these plants and identifying their mechanism of action.However, C. bonariensis, S. obtusifolia, W. ugandensis, and Z. usambarense use in snakebite is limited by poor efficacy and cytotoxicity.

TABLE 2
The cytotoxicity of antivenom, extracts, and fractions of Commiphora africana, Vernonia glabra, and Zanthoxylum usambarense in Artemia salina.
50 , Lethal concentration of the test substance responsible for the death of 50% of Artemia salina larvae; µg/mL; Micrograms per millilitre; CI, confidence interval.Frontiers in Pharmacology frontiersin.org

TABLE 3
Qualitative phytochemical composition of the extracts and fractions of Commiphora africana and Vernonia glabra.