Investigation of anaesthetic potentials of various extracts of Annona muricata (sour sop) in Wister albino rat and dog

1 Introduction

The need for efficacious, safe local and general anaesthetics is of paramount importance in surgery. There is no absolutely safe local and general anaesthetic in surgical practice (Neal et al., 2010) leading to continuous quest for anaesthetics with relatively less toxicity and high effeicacy. Annona muricata (sour sop) is a member of Annonaceae family. Other examples of the species are Annona reticulata (Custard apple), A. squamosa (Sugar apple) and A. cherimosa. Unripe roots, seeds, leaves and fruits of the plant are used as insecticides, pesticides and insect repellants respectively (Moghadamtousi et al. 2015). Annona muricata contains neurotoxic long-chain fatty acids called acetogenins and annonacin which acts via mitochondria by inhibiting nicotinamide adenosine dinucleotide reductase (NADH) and ubiquinone oxidoreductase. Alkaloids such as isoquinoline (reticuline), aporphines and protoberberine (coreximine), phenols (quercetin and gallic acids) and sesquiterpenes (Coria-Téllez et al., 2016), annonaine, nomuciferine and asimilobine have been isolated from Annona muricata (Hasrat et al., 1997a,b). Seeds, leaves and pericarp have acetogenin (annonacin), inhibitor of mitochondrial complex L inhibitor that induces nigral and striated neurodegeneration in rats (Escobar-Khondiker et al., 2007). The leaves have anticonvulsant, anti-inflammatory, antinociceptive and analgesic effects via pathway of opioid receptors. Antioxidant, adaptogenic (Hamid et al. 2002) antihypertensive via calcium antagonism (Coria-Téllez et al., 2016 and wound healing via up-regulation of Hsp 70 have been reported (Moghadamtousi et al., 2015). The fruit can cause atypical Parkinsonism among French West Indians (Caparros-Lefebvre and Elbaz, 1999).

The flavor components of Annona muricata are methylhexanoate and methyl hex-2-enoate (MacLeod and Pieris, 1981). Hydroalcoholic and chloroform extraction could yield phytochemical principles with hypotensive effect. Such compounds have alkane, ketones and alcohol functional groups as demonstrated by Hibiscus rosasinensis Linn (Siddiqui et al., 2005). Water and organic solvents could increase the yield of some phytochemical principles (Sunday et al., 2019). Therefore, local and general anesthetic potentials of various extracts of Annona muricata were studied in rat and dog respectively with intent to discovering highly efficacious, safe anaesthetic from the plant.

2 Materials and methods

3 Methods

4 Results

Qualitative and quantitative analyses revealed presence of saponins, flavonoids, tannins, alkaloids, glycosides and phenols in the leaf, stem bark, root, root bark and stem but anthraquinone was absent (Tables 1 and 2). The results of limit dose test of 5000 mg/kg are presented in Table 3. No male rat died throughout the period of experimentation, but all the three female rats died at the same dose level. The observed toxicity signs are pruning of hair; roaming, standing still, calmness, sedation for 3–5 min, male mounting another male, lateral and ventral recumbency and anaesthesia were observed. The scoring of pain sensation to assess local anaesthetic potential of Annona muricata aqueous leaf extract is presented in Table 4. Loss of pain sensation that lasted for > 1 h was significantly higher (p < 0.05) at all the four anatomical sites: tail, linea alba, left paralumbar and right paralumbar regions in comparison with the pre-treatment sensation test. The vital parameters and clinical signs observed from the puppies administered various doses of Annona muricata extracts are presented in Table 5. It was only puppy administered 170 mg/kg of stem bark water extracted that produced a complete state of general anaesthesia that lasted for 1 h 07 min. The signs observed are respiratory rate (32 cycles per min), heart rate (60 beats per min), open eye sedation for about 3 min, full anaestesia similar to that of plane 3, stage 3, deep respiration, salivation which disappeared after 5 min, defecation and urination. The lengths of small and large intestines measured are 220 cm and 32 cm, respectively. The values of vital parameters measured after surgery were heart rate (60 beats per min) and respiratory rate (70 cycles per min), respectively (Table 5). The surgery was successfully done (Figs. 1–4). However, the animal died of bleeding the following day, suggesting haemolytic potential of the extract.Table 5.

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Fig. 1

Mongrel dog treated with 170 mg/kg of Annona muricata stem bark water extract in the state of open eye anaesthesia.

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Fig. 2

Mongrel dog anaesthetized with 170 mg/kg of Annona muricata stem bark water extract undergoing laparotomy for measurement of large and small intestines.

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Fig. 3

Mongrel dog anaesthetized with 170 mg/kg of Annona muricata stem bark water extract in draping.

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Fig. 4

Mongrel administered 170 mg/kg of Annona muricata stem bark aqueous extract recovering from general anaesthesia after laparatomy.

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5 Discussion

High quantities of secondary metabolites present in the plant suggest likely vast number of biological activities of the plant. The survival of three male rats administered 5000 mg/kg body weight of Annona muricata bark root methanol extract shows that the LD₅₀ of the extract is above 5000 mg/kg. The death of three female rats administered 5000 mg/kg of the extract shows that the female rats are more sensitive to the extract than the male rats are. Our finding agrees with the report indicating that plant materials could be toxic and their toxicity depends on the dose, phytochemical principle, sex, species and age of animals (Saganuwan 2016). However, our finding disagrees with the report indicating that the LD₅₀ of A. muricata leaf extract was 1091.7 mg/kg body weight (Coria-Téllez et al., 2018). The difference in the LD₅₀s may be due to differences in solvents used, part of plant tested and soil chemistry on which the plants are cultivated. The muscle relaxation, local anaesthetic and sedative effects of aqueous leaf and root bark methanol extract respectively observed on male and female dogs show that the plant is a source of potent anaesthetic. Gonzalez-Trujano et al. (2001) had reported that the plant has anticonvulsant activity. Palmitone (16- hentriacontanine) isolated from the leaves of Annona muricata diversifolia showed anticonvulsant activity on pentylenetetrazole- induced clonic-tonic seizures. It delayed the onset of seizure, reduced death caused by 4- amino pyridine (4-AP) and bicuculine. However, palmitone didn’t produce motor incoordination and loss of righting reflex used as signs of neurological impairment. A dose of palmitone (1.85 mg/kg) was more potent than etosuximide (59.6 mg/kg), sodium valproate (63 mg/kg), carbamazepine (>300 mg/kg) and four folds less potent than diazepam (0.48 mg/kg), respectively (N’gouemo et al., 1997). The mechanisms of action may be via gamma aminobutyric acid (GABA) receptors (Gonzalez-Trujano et al. 2001). Single repeated administration of clozapine on phencyclidine-induced hyper locomotion, down regulated 5-HT (2A) receptors and blocked the enhanced phencyclidine-induced neurochemical and behavioural changes (Abekawa et al. 2007). The general anaesthesia shown by the male dog administered 170 mg iv (Figs. 1–4) indicates that the extract has general anaesthetic principle. General anaesthesia (hypnosis, muscle relaxation and analgesia) is a state of unconsciousness in which the animal no longer responds to innate instinct so as to avoid vulnerability of dorsal recumbency known as loss of righting reflex (LORR). The end-state is marked by return of the righting reflex (RORR). Therefore, hypnosis in animals is less straight forward than it is in human (McCarren et al. 2013). Because of neurotoxic tendency of sour sop, it can cause neurodegenerative, atypical Parkinsonism (Caparros-Lefebvre and Elbaz, 1999). Reticuline from the plant inhibits dopamine uptake, at high concentration, it is toxic to GABAergic and dopaminergic receptors. It is a weak neuromuscular (nicotinic) calcium channel blocker, causes uterine relaxation and vasorelaxation activity via l-type Ca²⁺ channel. The analgesic effect of Annona muricata is via opioidergic pathway and anti-inflammatory effect is via inhibition of chemical inflammation mediators (Ishola et al. 2014). The low respiratory rate observed during the period of anaesthesia (Table 5) agrees with the report indicating that respiratory sinus arrhythmia could be used for prediction of slow wave brain activity during sleep (Niizeki & Saitoh 2018). Agents that cause deep sleep (hypnosis) are GABA agonists and many other CNS depressants by altering physical properties of lipid in neuronal membranes (Mendelson 2002). Injectable anaesthetic can be used for the induction and maintenance of short-term anaesthesia that prevents detrimental effects of pain by establishing preemptive and multimodal analgesia (Sullivan et al. 2016). The salivation and open eye sleep observed in the present study agrees with the report indicating that some general anaesthetics have salivation as one of the side effects. Dissociative anaesthetics including ketamine have salivation and open eye sleep as side effects (Jud et al.2010). General anaesthesia is induced by intravenous drug and maintained by inhalational anaesthetics. If anaesthesia is maintained by intravenous anaesthetic, autonomic function remains more stable intraoperatively. Hence hypnosis, analgesia and muscle relaxation may improve the welfare of animal undergoing general anaesthesia (Tajeda-Chavez et al. 2018) as observed in the present study. The ability of the extract to cause full general anaesthesia using a single large bolus to fill the volume of distribution of the central compartment, disagrees with the report indicating that the technique of intravenous anaesthesia requires lower dosages of anaesthetic after large volume, to maintain effective drug plasma concentration for the duration of anaesthesia (Waelbers et al. 2009).

Annona is used for treatment of insomnia, convulsion, anxiety, and causes sedation, parturition, milk let down and spasms. Annonaine and asimilobine (aporphines) are antidepressants whereas coreximine (protoberberine) and reticuline (isoquinoline) are neurotoxic. The mechanism of action of annonaine is via dopamine inhibition. Also, nornuciferine (isoquinoline) has antidepressants activity. The neuroactive principles are present in fruit, leaf, root and stem, but annonacin has neurodegenerative effect (Moghadamtousi et al. 2015). Behavioural and neurological effects of A. muricata were via gamma aminobutyric and monoaminergic receptors. The purified plant extracts could be source of a new compound with local and general anaesthetic potential (Souza et al. 2018). The first anaesthetic discovered from plant, Erythroxylon coca is cocaine (Biondich and Joslin, 2016). Alkaloids and flavonoids have anaesthetic effects (Udegbunam et al. 2012). Cocaine isolated from E. coca is the prototype of local anaesthetics. Thymol and eugenol from Thymus vulgaris and Syzygium aromaticum are mechanistically and structurally similar to intravenous anaesthetic of phenol origin. The anaesthetic phytochemicals act via gamma aminobutyric acid type A receptor, sodium channel, lipid membranes and N-methyl-d-aspartate receptors (Tsuchiya, 2017). The nociceptive impulses are blocked, providing analgesia with minimal risk of adverse effects (Grubb and Lobprise, 2020). The depressant activity of chemical compounds depends on their functional groups and structure–activity relationship (Saganuwan, 2017a,b). The bleeding observed in the present study agrees with the report indicating that many herbal anaesthetic agents cause coagulation, endocrine disruption, cardiovascular problems, hepatotoxicity and prolongation of anaesthetic effects (Cheng et al. 2002). Kaempferol, apigenin, benzoylaconine, aconitine, chalcone, catechin,valtrate, pyrethrin and beta-carboline have anaesthetic and pain relief potential (Sayhan et al., 2017). Hence the anaesthetic potential of medicinal plants depends on the plant parts, solvents and the innervations of injected anatomical sites (Lopez et al. 2016).

Central nervous system disorders could be treated or managed by chemical agents that modify the tight junction for easy transport to the brain (Liu 2008). The reaction may be redox at the level of the lipophylic membrane and it is dependent on molecular weight, pH, physicochemical and pathological condition of blood–brain barrier. Some CNS acting agents may undergo carboxylation, carbonylation, methylation, desulphation and dehydrogenation with low molecular weight (Saganuwan, 2017a,b). But carotid bodies comprise glomus cells and sustentacular cells, sense pH, partial pressure of oxygen, carbondioxide and glucose. Sensory information is sent to brainstem neurons for regulation of respiratory airway ventilation, circulatory and endocrine responses (Saganuwan 2019). Death of the operated dog could be attributed to blood loss observed after surgery. Hence concurrent use of antihaemolytic with the extract may be evident.

6 Conclusion

Annona muricata root bark methanol extract is more toxic in female rats as compared to male rats. A dose of 12.5 mg/kg of the leaf extract caused significant local anaesthesia. However 1250 mg/kg caused deep anaesthesia in rat and was translated to 170 mg/kg that caused deep anaesthesia that lasted for 67 min and permitted laparotomy in dog. Hence, A. muricata may serve as source of less toxic and high efficacious local and general anaesthetic that may provide balanced anaesthesia comprising analgesia, deep hypnosis and muscle relaxation (triad of anaesthesia).