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Byline: M. Farha, L. Parkianathan, N.A.I. Abdul Amir, V. Sabaratnam and K.-H. Wong

Keywords: Lignosus rhinocerotis; aqueous extract; peripheral nerve regeneration; functional recovery; neurotoxicity


Peripheral nerve injury always results in restricted activity or life-long disability. Traumatic peripheral nerve injury is caused by motor vehicle accidents, falls or penetrating injury. It leads to disruption of intraneural circulation. Sciatic nerve of the lower limb is most often injured followed by peroneal, femoral and tibial nerves. Symptoms experienced would be the numbness of the injured limb, intolerance towards cold, neuropathic pain and in severe cases, paralysis (Grinsell and Keating, 2014). Although microsurgical treatments for nerve injuries have been improved over the past decades, the outcome of peripheral nerve injury repair remains unsatisfactory (Popa et al., 2016). Pre-clinical animal models such as rat, mouse, rabbit, dog, cat, sheep, monkey and pig have been employed for evaluation of peripheral nerve regeneration and repair prior to implementation in clinical practice (Angius et al., 2012).

Mecobalamin, a vitamin B12 derivative is a commonly prescribed medication for peripheral nerve disorders. However, it is associated with side effects such as urticaria, eczematous, exanthematous skin lesions, anaphylactic reactions, polycythemia with peripheral vascular thrombosis, and movement disorders with tremor and myoclonus (Aronson, 2016). Therefore, much emphasis is placed on the potential of using natural resources as neuroprotective and neurotrophic agents (Seow et al., 2013). Natural products that stimulate neurite outgrowth and may substitute nerve growth factor (NGF) or exhibit NGF-like activities that cause neurons and myelin to regrow have been extensively studied. In Malaysia, Lignosus rhinocerotis (Cooke) Ryvarden or Tiger's milk mushroom is hailed as national treasure and has been consumed by the indigenous communities as mycomedicine for multiple diseases. The sclerotium of the mushroom is greyish brown in colour with irregular hard mass appearance.

Sclerotial extracts were reported to elicit immunomodulatory effects and tumor suppressor activity (Wong et al., 2009a; Wong et al., 2011a), possessed neuritogenic effects in PC12 cells (Seow et al., 2013) and dissociated cells of brain, spinal cord and retinal cells (Samberkar et al., 2015), and anti-neuroinflammatory effects in BV2 microglia (Nallathamby et al., 2016; Seow et al., 2017). In addition, Jhou et al. (2017) demonstrated that L. rhinocerotis was not teratogenic. Subacute toxicity assessment revealed that L. rhinocerotis did not have adverse effects on visceral organs namely the liver, kidney, heart, spleen and lungs (Lee et al., 2011). However, there is no information on neurotoxicity in in vivo studies. Due to the broad range of ethnomedicinal properties of L. rhinocerotis, the neurotoxicity data is vital before it could be developed as a functional food (van der Heijden et al., 1999).

Peripheral Nervous System has an intrinsic growth capacity for repair and regeneration. Therefore it is a widely accepted model for neuroregenerative research (Huebner and Strittmatter, 2009). Based on these promising in vitro neuroprotective effects, we investigated the neuroregenerative properties of locally cultivated L. rhinocerotis aqueous extract on functional recovery following crush injury to the sciatic nerve in Sprague-Dawley rats and examined the subacute toxicity (if any) of the extract on Peripheral and Central Nervous Tissues.


The study was carried out at Department of Anatomy, Faculty of Medicine, University of Malaya, Kuala Lumpur in 2017.

Preparation of L. rhinocerotis aqueous extract: The freeze-dried cultivated sclerotium of L. rhinocerotis marketed as Ligno TM02 (Batch No. PL/16/014-1215) was purchased from Ligno Biotech Sdn Bhd, Selangor, Malaysia. Aqueous extraction was done according to Seow et al. (2015). The L. rhinocerotis sclerotium powder was weighed and mixed in distilled water with a sample-to-water ratio of 1:10 (w/v). The mixture was agitated in the shaker at 50 rpm at 45AdegC for 24 h. Following incubation, the mixture was double boiled in a 100AdegC water bath for 30 min. After cooling to room temperature, the mixture was centrifuged at 10 000 rpm at 4AdegC for 30 min. Supernatants were freeze dried at -50AdegC for 48 h (Wong et al., 2009b). The resultant fluffy white product was stored in a -20AdegC freezer prior to further use.

Principles of rat grouping: The in vivo study was done with approval by Institutional Animal Care and Use Committee, Faculty of Medicine, University of Malaya (Ethics Ref No 2013-10-08/ANAT/R/WKH). Twenty-four adult female Sprague-Dawley rats with body weight in the range of 180 g to 200 g were randomly assigned to four groups of six rats each; (i) negative control that received distilled water (5 ml/kg body weight/day), (ii) positive control that received 130 ug mecobalamin/kg body weight/day (MethycobalA(r), 500 ug vitamin B12 in each tablet, Eisai Company Limited, Japan) (iii) low dosage that received 500 mg of L. rhinocerotis aqueous extract/kg body weight/day and (iv) high dosage that received 1000 mg of L. rhinocerotis aqueous extract/kg body weight/day. All the rats underwent 14 days pre-treatment prior to sciatic nerve injury. The oral administration was continued for the next 28 days after injury.

Observations were recorded from day 0 (1 h before injury) and at every 4 days interval until day 28. Food pellet and drinking water were available ad-libitum.

Surgical procedure: Rats were injected intraperitoneally with a mixture of ketamine (50 mg/kg) and xylazine (5 mg/kg). Incision on skin and gluteal muscle was made by a surgical blade no. 4 (Wong et al., 2015). The sciatic nerve was pinched 1 cm from the bifurcation point by a watchmaker forceps no. 4 for 10 s. Complete crush was indicated by a translucent band on the nerve. The muscle and skin were sutured using a non-absorbable suture and antiseptic cream was applied thereafter. All operations were performed on right limb and the left limb served as an uninjured control.

Evaluation of functional recovery: Vibrissae of the rats were observed and ensured to be actively moving. Actively moving vibrissae indicates the readiness of rat for the study (Prescott et al., 2011).

i. Sensory function: Hot plate is generally utilized for estimating sensitivity to thermal pain. The hot plate with an open-ended cylindrical space (Panlab, S.L. Spain) was heated to 50AdegC and the rats were placed on the plate. This study explores behavioural components, namely the paw licking and jumping that are associated with their reaction times. The time taken to elicit a withdrawal response of injured hind limb from the hot plate was recorded by a built-in timer with an external foot switch and termed as withdrawal reflex latency (WRL) (Wong et al., 2015). WRLs were recorded before injury (day 0) and after 4, 8, 12, 16, 20, 24 and 28 of injury. The injured hind limb was tested 3 times and averaged to obtain mean WRL. Non-responsive rat was removed from the hot plate after 30 s of heat stimulation to prevent tissue damage, and therefore was given WRL of 30 s.

ii. Motor function: The rats were inspected after 4, 8, 12, 16, 20, 24 and 28 of injury. The toe-spreading reflex was observed by lifting the rat at its tail, the hind limbs were hanging free and forelimbs were resting on the workbench. Each rat was observed for 1-2 min and activities were categorized according to the toe-spreading reflex of injured right hind limb: 0 - no spreading; 1 - minimal spreading; 2 - average spreading; 3 - normal spreading (Wong et al., 2009b).

Histopathological analysis of nervous tissues: After 28 days of injury, Peripheral Nervous Tissues (sciatic nerve and dorsal root ganglia) and Central Nervous Tissues (cerebrum, cerebellum and spinal cord) were harvested and fixed in Davidson's solution for 24 h followed by 10% neutral buffered formalin solution. The tissues were processed and stained with hematoxylin and eosin. Specimens were examined microscopically for the evidence of toxicity under a light microscope. Toxicological changes in nervous tissues such as neuronal degeneration and swelling or apoptosis of neurons were examined.

Statistical analysis: The means of data (n=6 animals per group) were subjected to one-way analysis of variance (ANOVA) and the significance of the difference between means was determined by the Duncan's multiple range tests (DMRT) at 95% least significant difference (p a$? 0.05).


Following crush injury, there were no signs of self-mutilation. Dragging of feet and clumping of toes of the right hind limb were observed (Fig. 1). The efficacy of the treatments on the sensory and motor function of the right hind limb was compared to the uninjured left hind limb. Hot plate test was used to evaluate the sensory functional recovery of the rats. Rats which had the nociceptive sensitisation responded by (i) hind limb withdrawal, (ii) hind paw licking or (iii) jumping off the hot plate. Prior to sciatic nerve injury, the rats responded to heat stimulus within 30 s. Fig. 2 shows the data for the WRL over the period of 4 weeks. Rats did not respond to heat stimulus in the first week following sciatic nerve crush indicating loss of temperature and pain sensation on the right foot. Withdrawal response began on day 8 after crush injury and towards the end of week 3 in low dosage and negative control groups, respectively (p a$? 0.05).

WRL was then improved during the 4-week recovery time. Mecobalamin and high dosage groups did not show any improvement in WRL throughout the recovery period. WRLs of the low dosage-treated rats were significantly less than the mecobalamin group from day 12 onwards (p a$? 0.05). Low dosage group recovered better compared to mecobalamin group (p a$? 0.05). There was a significant difference between rats in mecobalamin and negative control groups from day 24 onwards (p a$? 0.05). WRLs in low dosage group did not reach statistically significant difference from other groups after 4 and 8 days of injury.

Fig. 3 shows the toe-spreading reflex degree. Maximal toe-spreading was recorded in low dosage group on day 12 (2 rats), 20 (1 rat), and 28 (1 rat) and in high dosage group on day 16 (1 rat), 24 (1 rat) and 28 (4 rats) after injury. However, rats in the negative control and mecobalamin groups failed to achieve maximal toe-spreading on day 28 after injury. After 2 weeks of injury, the first signs of functional motor recovery were noticeable. Toe-spreading precedes recovery of sensory function in high dosage group following sciatic nerve crush injury. Jin et al. (2012) postulated that an early return of reflex is not a promising sign of nerve regeneration. It manifests an increased sensitivity of denervated muscles to adrenalin. Adrenalin released into systemic circulation during reflex-testing may cause muscle contraction mimicking nerve-induced toe-spreading reflex.

The gross examination of the nervous tissues of rats treated with L. rhinocerotis did not display any significant pathological changes compared to the negative control and mecobalamin groups after 28 days of injury. The microscopic sections of the Peripheral Nervous Tissues namely, the sciatic nerve and dorsal root ganglion and the Central Nervous Tissues namely, the cerebrum, cerebellum, spinal cord are illustrated in Fig. 4-8. In the controls and treated rats, the longitudinal sections of sciatic nerve show parallel bundles of nerve fibres encased by perineurium (Fig. 4) and dorsal root ganglion displayed normal microscopic appearance of cell bodies of sensory neurons surrounded by satellite cells (Fig. 5).

As for the Central Nervous Tissues, cerebral cortex of cerebrum consists of the largest pyramidal cells or Betz cells in layer V and cell bodies of other neurons (Fig. 6), cerebellar cortex of cerebellum consists of typical microscopic features of large flask-shaped Purkinje cells in middle layer, scattered basket cells in molecular layer and densely packed granular cells in granular layer (Fig. 7), whereas ventral horn of spinal cord consists of normal architecture of multipolar motor neurons (Fig. 8). Therefore, there was no abnormal changes in terms of neuronal degeneration, swelling or apoptosis was detected.


Nerve crush or axonotmesis is characterized by a disruptive lesion of the axon and its myelin sheath. The nerve segment distal to the injury site undergoes cellular alterations that characterise Wallerian degeneration. However, the connective tissue coverings namely the endoneurium, perineurium and epineurium remain intact to guide and redirect the growth of axonal buds during the regeneration process. Crush injury to the sciatic nerve causes impaired extension at the hip joint, impaired flexion at the knee joint, loss of dorsiflexion and plantar flexion at the ankle joint and loss of inversion and eversion of the foot (Drake et al., 2014). The rationale for the utilization of crush injury over transection is due to successful regeneration guided by Schwann cells and maintenance of endoneurial tubes that enhances axonal elongation and subsequently reinnervation of skeletal muscle fibres (Valero-Cabre et al., 2004).

Crush injuries are being employed in the studies of molecular events that participate in peripheral nerve regeneration. Withdrawal latency stimulated by nociception and cutaneous stimulation are functionally unadapted in neonatal rats (Miranda et al., 2006). The task-specific nociceptive withdrawal reflex organization then gradually emerges over the first three postnatal weeks. The reflex patterns in postnatal maturation reflects a tuning of spinal connectivity. Mecobalamin is widely used for the treatment of peripheral and diabetic neuropathies (Lawrence, 2016). Concentrations in the range of 100 to 130 ug/kg body weight were recommended in peripheral nerve crush regeneration study using rat sciatic nerve model (Jiang et al., 2009; Ma et al., 2010). In our study, mecobalamin at 130 ug/kg body weight did not improve functional recovery over the period of study, and therefore higher dosages than 130 ug/kg body weight warrant further testing.

Ultra-high dosage of mecobalamin at 500 ug/kg body weight has been reported to promote regeneration in a Wistar rat model of acrylamide neuropathy (Watanabe et al., 1994) and neuroprotection in a Wistar rat model of sciatic nerve ligation and crush injury (Muthal et al., 2008), whereas 30 mg/kg body weight has been found to delay the progression of motor and neuropathological symptoms in a Wobbler mouse model of amyotrophic lateral sclerosis (Ikeda et al., 2015). Compounds that exhibit neuroprotective and neuroregenerative activities are the cordycepin from Cordyceps militaris (Cheng et al., 2011), complex carbohydrates and sterols from Ganoderma lucidum (Zhao et al., 2005), lysophosphatidylethanolamine from Grifola frondosa (Nishina et al., 2006), phenol derivatives and diterpene xylosides from Hericium erinaceus (Kawagishi et al., 2008), alkaloids and diterpenes from L. rhinocerotis (Seow et al., 2015) and cerebrosides from Termitomyces albuminosus (Qu et al., 2012).

With these findings, it interests us to explore the effects of L. rhinocerotis on functional recovery by using an in vivo model of sciatic nerve crush injury. Lignosus rhinocerotis hot and cold aqueous extracts have been found to possess a broad spectrum of cytotoxic activities, immunodulatory, antimicrobial, antiviral, fibrinolytic and neuritogenic activities attributed to the fatty acids, polysaccharides, polysaccharide-protein complexes, glucans, peptides, proteases and proteins (Lau et al., 2015; Nallathamby et al., 2018). Cold aqueous extract was cytotoxic against human cell lines, namely the acute promyelocytic leukaemia, breast adenocarcinoma, colorectal carcinoma, prostate adenocarcinoma, lung carcinoma, lung fibroblast, hepatocellular carcinoma, embryonic liver, squamous carcinoma and nasopharyngeal carcinoma (Lau et al., 2013). Our results showed that daily oral administration of low dosage of L. rhinocerotis aqueous extract enhanced functional recovery of damaged sciatic nerve.

We revealed a neuroregenerative role of polysaccharide (Wong et al., 2015) and [beta]-glucan from H. erinaceus aqueous extract in the Peripheral Nervous System (Wong et al., 2016). Thus the polysaccharide component of L. rhinocerotis aqueous extract plays a role in its neuroregenerative effect. On the other hand, water-soluble polysaccharides of Tremella fuciformis has also been shown to trigger NGF production, and ameliorate memory and learning deficits (Kim et al., 2007). Low dosage of L. rhinocerotis aqueous extract increased the pain sensitivity to heat and enhanced the reflex response of injured right hind limb after 8 to 12 days of injury. These significant changes were observed subsequently during the study period of 28 days. Even when promising sensory recovery occurs, motor deficits may impair the functional outcome. Recovery of motor function depends on the integrity of skeletal muscle upon arrival of regenerating axons.

Reinnervation of denervated skeletal muscles occurs after 18 to 24 months of injury with the exception of hand intrinsic muscles at 6 months after injury. However, Ma et al. (2011) postulated that lacking of motor function after injury is due to failure of synapse or formation of neuromuscular junction after extended denervation rather than attenuation of axonal growth. Further, a functional motor performance depends on the interactions between primary motor cortices or inter-hemispheric functional coupling (Kajal et al., 2017). Studies have also shown that peripheral nerve injury produces neuropathic pain and memory deficits in the hippocampal CA3 area (Joshi et al., 2017), parahippocampal cortex, posterior hippocampus, retrosplenial cortex, angular gyrus, posterior cingulate cortex and precuneus (Robin et al., 2018). Brain-derived neurotrophic factor (BDNF) plays a crucial role in regulating synaptic plasticity and neuronal activity.

Histone deacetylase inhibition in the aged brain has been shown to improve spatial memory in a BDNF-dependent manner (Snigdha et al., 2016). Multiple lines of evidence have documented that free radicals generated after tissue injury delay functional recovery. However, antioxidants and anti-inflammatory agents that reduce ischemic injury accelerates functional recovery (Algora et al., 1996). Owing to this, L. rhinocerotis has been shown to possess anti-inflammatory (Lee et al., 2014; Nallathamby et al., 2016), neuritogenic (Seow et al., 2013) and free radical scavenging (Lau et al., 2015) activities and therefore could relieve neuropathic pain and multiple effects of peripheral nerve injury. Generally, the issue of low bioavailability requires the use of very high dosages to obtain therapeutic effects in vivo and therefore are more likely to cause side effects (Caillaud et al., 2018).

Although high dosage of aqueous extract at 1000 mg/kg body weight/day did not produce any signs of toxicity or mortality (as discussed in the following sections), it did not improve WRL and had minimal effect on the return of toe-spreading over the period of study. Administration of high concentration of undesirable substances that may also act as inhibitory substances can mask the effect of the actual components such as polysaccharides or [beta]-glucans responsible for functional recovery enhancement (Turner et al., 2011). Our observation is in accordance with several studies in animal models. Morus alba aqueous extract at 0.1 mg/kg improved motor and sensory functions after crush injury. Increased dosages of 1.0 and 10 mg/kg failed to show any significant recovery (Mucimapura et al., 2010). Huang et al. (2011) revealed that low dosage of Paeoniae alba Radix water extract at 1.25 and 12.5 mg/ml in silicone rubber chambers promoted nerve fibres to grow across a 10-mm gap lesion in sciatic nerve.

The effect was reversed by a high dosage of 125 mg/ml. Chang et al. (2011) demonstrated a nerve growth-suppressing action by high dosages of earthworm extract at 250 to 1000 mg/ml. Further, local infusion of low-dosage curcumin at the injury site at 0.2 mg/day for 4 weeks has been demonstrated to improve functional recovery, nerve conduction velocity and remyelination after sciatic nerve injury (Caillaud et al., 2018). Therefore, an optimum dosage of L. rhinocerotis aqueous extract should be determined to achieve maximal potential of peripheral nerve regeneration. In other words, there are threshold dosages above and below a certain effective dose. Various studies on peripheral nerve regeneration by natural products are being actively conducted worldwide. Buyang Huanwu decoction promoted regeneration of a 10-mm gap formed between the proximal and distal stumps of rat sciatic nerve (Cheng et al., 2001).

Jiang et al. (2009) verified that chitooligosaccharides enhanced sciatic nerve regeneration in a rat model of crush injury through functional and morphological assessment. Ding et al. (2008) reported the therapeutic effect of Achyranthes bidentata root aqueous extract in accelerating nerve regeneration in a rabbit model of crushed common peroneal nerve. The polypeptides of aqueous extract has also been shown to enhance peripheral nerve regeneration and functional recovery after crush injury in mice (Yuan et al., 2010), rats (Wang et al., 2013) and rabbits (Cheng et al., 2014). Astragaloside IV (AS-IV), a purified compound of Radix astragali has been found to accelerate remyelination in a mice model of sciatic nerve injury. The effect was modulated by upregulation of GAP-43 (Lu et al., 2010). In addition, Ramli et al. (2017) had reported the efficacy of evening primrose oil at 6000 mg/day in accelerating the rate of sciatic nerve regeneration after crush injury in rat.

Wong et al. (2011b; 2015; 2016) explored the role of local protein synthesis in regenerating axons, combined acetylcholinesterase and silver method for demonstrating motor endplates, and expression of Akt, MAPK, c-Jun and c-Fos in the dorsal root ganglia in accelerating motor and sensory functional recovery after crush injury by H. erinaceus. The neurotoxicity test of L. rhinocerotis is crucial before developing it as a functional food. Several substances have been reported to interfere with functional recovery. Greensmith et al. (1994) postulated that N-methyl-D-aspartate, a glutamate agonist in developing spinal cord has resulted in apoptosis of motor neuron following sciatic nerve injury during postnatal period but not in adult motor neurons. Montanez et al. (2000) revealed an interaction between seizures and anticonvulsants administered simultaneously following a brain injury.

Application of phenobarbital before initiation of subclinical seizures was found to be more destructive in the process of recovery than seizures alone, or treatment with phenobarbital after the seizures. The impact depends on timing of phenobarbital administration, its mechanism of action and frequency of seizures. In a study by Nakamura et al. (2001), intravenous administration of vincristine, a chemotherapy medication has been shown to cause a dose-dependent delay in functional recovery in a mouse model of sciatic nerve crush although weight loss or disruption of grooming and social behaviour were not observed throughout the 4-week experimental period. Generally, medicinal mushrooms exhibit minimal adverse effects compared to the drugs, nevertheless scientific toxicity data is required.

Pertaining to the previous studies on the toxic effect of L. rhinocerotis, lyophilized mycelium up to 3400 mg/kg did not cause either mutagenicity in an in vivo ICR mice model of micronucleus assay (Chen et al., 2013) nor did it provoke toxicity in dams or teratogenicity in fetal development during the critical period of embryonic organogenesis (Jhou et al., 2017) Further, in a subacute toxicity test for 28 days, oral administration of two cultivars of L. rhinocerotis, namely TM02 in the range of 250 to 1000 mg/kg and TM03 at 1000 mg/kg, and wild type at 1000 mg/kg did not produce any signs of toxicity in adult rats as confirmed by haematological analysis, clinical biochemistry, urinalysis and histological analysis of vital organs (Lee et al., 2011). Lee et al. (2013) extended their investigations into the chronic toxicity of cultivar TM02. Administration of sclerotial powder over the same range of dosages for 180 days did not show adverse effects.

In addition, sclerotial powder administered at 100 mg/kg over the period of 7-8 weeks also did not reveal fertility disorders nor causing teratogenic effect in the offsprings. Our toxicity study on nervous tissues following subacute oral administration of L. rhinocerotis showed no adverse effects. The microscopic evaluation of the nervous tissues treated with aqueous extracts did not show any difference in histological structure compared to the negative control and mecobalamin. The gross examination showed no changes in the architecture of sciatic nerve, dorsal root ganglion, cerebrum, cerebellum, and spinal cord. Parameters indicating signs of toxicity were not observed. Therefore, this confirms the safety profile of L. rhinocerotis. These dosages can be further tested in human trials for the development as functional food.

Conclusion: The oral administration of L. rhinocerotis aqueous extract at 500 mg/kg body weight/day has been demonstrated to enhance functional recovery following peripheral nerve injury. Dosages at 500 and 1000 mg/kg body weight/day had no toxicity effects on nervous tissues. Therefore it could be developed as a functional food with minimal adverse effects.

Acknowledgements: Fundamental Research Grant Scheme FP011-2016 from the Ministry of Higher Education Malaysia and University of Malaya High Impact Research Grant (UM.C/625/1/HIR/MOE/ASH/01) from the Ministry of Education Malaysia.


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Publication:Journal of Animal and Plant Sciences
Date:Aug 27, 2019

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