Ethanolic extract of Aloe vera ameliorates sciatic nerve ligation induced neuropathic pain.

BACKGROUND: Aloe vera is being used since ages by human kind for treating various ailments including various inflammatory conditions, but scientific validation has not been done for analgesic activity against neuropathic pain.
OBJECTIVE: The current study was designed to systematically evaluate the therapeutic potential of the ethanolic extract of A. vera (EEAV) against sciatic nerve ligation (SCNL) induced neuropathic pain.
MATERIALS AND METHODS: Nociceptive threshold of EEAV against thermal hyperalgesia, chemical hyperalgesia and mechanical allodynia were performed on 0, 7, 14 and 21(st) day post-SCNL. Serum total protein, serum nitrite, in vivo anti-oxidant parameters and lipid peroxidation (LPO) were estimated. Sciatic nerve homogenate was used to estimate myeloperoxidase (MPO) and calcium levels. Histopathology of the sciatic nerve was done to confirm the biochemical findings.
RESULTS: Treatment with ethanolic extract has increased the threshold for the nociception in thermal hyperalgesia, chemical hyperalgesia and mechanical allodynia models. A significant improvement of in vivo anti-oxidant parameters and decreased LPO levels were observed on treatment with A. vera. Significant decrease in serum nitrite, protein, calcium and MPO levels were observed, indicating protection against damage caused by SCNL.
CONCLUSION: The results of the present study validate the use of EEAV to treat neuropathic pain. This effect may be attributed to the decreased migration of neutrophils and due to the anti-oxidant properties of A. vera. Further studies to confirm the mechanism of action will help develop suitable A. vera formulations for neuropathic pain therapy.

INTRODUCTION

Neuropathic pain is “pain arising as a direct consequence of a lesion or disease affecting the somatosensory system.” It is a common clinical problem affecting millions of people around the world and difficult to manage with the current conventional antinociceptive treatments due to the poor efficacy and associated adverse effects. The search for new drug molecules to alleviate pains of this sort is a priority nowadays. Elucidating the molecular mechanisms of neuropathic pain is an important prerequisite for the rational development of novel analgesic drugs for the therapy of chronic pain.[1]

Aloe vera (Family: Liliaceae) is very commonly used in traditional Chinese and Ayurvedic medicine. It is called as “ghrtakumari” in Ayurvedic texts and is known as a cathartic. Among the 150 reported active constituents of Aloe, 12 anthraquinones and a phenolic compound having immunostimulating effects and antibiotic properties were extensively studied.[2] In Ayurveda, A. vera was also used as a remedy for cancer for which molecular mechanisms of action were considerably reported. A. vera acts on different signaling molecules like nuclear factor kappa B, Her2/Neu, caspases and matrix metalloproteinases.[3]

Acemannan, the active constituent of A. vera is a linear polysaccharide composed of (1,4)-linked mannosyl residues, with C2 or C3 acetylated, and some side-chains formed by galactose units attached to C6. Acemannan is known for its ability to restore and boost the immune system by stimulating the production of macrophages, interferons and interleukins which help to destroy viruses, bacteria, and tumor cells. For instance, the anti-tumor activity of acemannan was also widely reported.[2]

The β-(1-4)-glycosidic bond configuration of acemannan is an important consideration in terms of the therapeutic effects of A. vera gel, since humans lack the ability to break enzymatically down these bonds. In particular, acemannan through its immunomodulatory function also acts as potent anti-inflammatory and analgesic agent.[4]

The anti-inflammatory and analgesic activity of A. vera preparations was considered to be due to the presence of water insoluble polysaccharides like acemannan, but no scientific work has been yet reported to explore or support their use in neuropathic pain. Hence, the aim of the present study was to evaluate the potential of ethanolic extract of A. vera (EEAV) against sciatic nerve ligation (SCNL) induced neuropathic pain.

MATERIALS AND METHODS

Animals

Adult female albino rats of Wistar strain weighing about (200–250 g) were purchased from Raghavendra Enterprises, Bengaluru. The animals were housed under standard conditions of (22 ± 2°C) 12-h light/12-h dark cycle with free access to standard rat pellet diet procured from Pranav Agro Pvt. Ltd., India and purified drinking water ad libitum. All experiments and protocols described in the present study were approved by the Institutional Animal Ethical Committee of Sri Padmavathi School of Pharmacy (CPCSEA NO: 1016/a/06//010/2012).

Chemicals

Ethylene diamine tetra acetic acid, epinephrine bitartarate, hydrogen peroxide, trichloroacetic acid, 5,5-dithiobis (2-nitrobenzoic acid), thiobarbituric acid, Griess reagent, hexadecyl trimethyl ammonium bromide (HETAB) and O-dianisidine were procured from Sigma Aldrich, India. Calcium and total protein assay kits were procured from Autospan Diagnostics Ltd., India.

Instruments

UV-visible spectrophotometer (Analytical systems, Model No: AUV 2060), electronic balance (Shimadzu, Model No: DS-852J), tissue homogenizer (Ever shine, Model No: 607), semi auto analyzer (Mispa excel, Version: 1.4e) and cooling centrifuge (Remi Model No: C24 BL) were used for the estimation of parameters.

Preparation of plant extract

The leaves of A. vera were collected in and around Sri Padmavathi School of Pharmacy during the month of November, 2011 and authenticated by Dr. B. Seetharam, Professor, Sri Venkateswara Ayurvedic Medical College, Tirupathi. A voucher specimen of the same has been deposited in stores of Sri Padmavathi School of Pharmacy.

The extraction of A. vera was done by solvent extraction method. In this, about 500 g of A. vera gel fillets were filled in a round bottomed flask and then extracted with 95% ethanol using reflex condenser over a water bath for 3 h. This process was continued for 3 cycles. The extract was evaporated using rotary flash evaporator and the EEAV so obtained was stored in air tight borosil screw cap vials (% yield - 15% w/w). Phenol-sulfuric acid test was done to identify the proportion of ethanol soluble carbohydrates present in the EEAV by the procedure given by Balasubramanian and Sadasivam.[5] The EEAV was suspended in water and administered to the experimental animals daily, as per the dose required. The neuroprotective activity of A. vera extract was studied at doses of 100 and 300 mg/kg, p.o., as many earlier studies have documented the safety of EEAV at the tested doses of the present protocol.[6]

Induction of neuropathic pain by sciatic nerve ligation

Peripheral neuropathy was induced by SCNL in rats. The model of SCNL is one of the most commonly used models because it is reliable and easily reproducible. In brief, female Wistar rats were deeply anesthetized with ketamine (50 mg/kg, i.m.) and xylazine (5 mg/kg, i.m.). The hairs on the lower back and thigh of the rats were shaved. The skin of the lateral surface of the right thigh was incised, and a cut was made directly through the biceps. The sciatic nerve was exposed, and two ligatures (silk sutures) were placed around the nerve tightly until small degree of constriction is visually observed in gastrocnemius muscle. After performing the ligation, muscular and skin layer was sutured in layers with catgut and suturing thread respectively. Sham operation was performed in eight rats by simple cut on the thigh skin and muscle followed by closure of the cut with sutures. Following this, the animals were allowed to recover with supply of food and water ad libitum.

Treatment schedule for evaluation of ethanolic extract of A. vera against neuropathic pain

The animals were grouped 48 h postsurgery into five groups (n = 8) and were treated for 21 days. The first group, Group I consisted of sham operated animals to serve as normal control. Group II consisted of animals recovering after SCNL and receiving vehicle alone to serve as disease control. Group III included SCNL animals treated with gabapentin (9 mg/kg, p.o.), for standard comparisons. Groups IV and V consisted of SCNL animals administered with EEAV (100 and 300 mg/kg, p.o.) for assessing the effects of EEAV in a dose-dependent manner. Nociceptive threshold in cold water immersion test, thermal hyperalgesia, cold and mechanical allodynia was assessed at weekly intervals on 0, 7, 14 and 21st day of treatment schedule as follows.

Thermal hyperalgesia (cold water immersion)

The paw withdrawal latency (PWL) to cold stimuli was recorded in seconds (with the help of a stopwatch) by submerging the hind paw of rats in cold water (4 ± 1°C). The baseline latency of paw withdrawal from cold water was established 3 times, 5 min apart, and averaged. The change in withdrawal latency of treated group and vehicle-treated control groups was compared.[7]

Thermal hyperalgesia (hot water immersion)

The development of thermal hypersensitivity associated with neuropathic pain was measured using mean PWL of the rat paw when dipped in a water bath maintained at 47 ± 0.5°C. The baseline latency of paw withdrawal from hot water was established similarly as mentioned in the cold water immersion method. A cut-off time of 15 s was imposed to avoid injury to the paw. The change in the PWL as compared to basal responses was calculated as a measure of hyperalgesia in all the groups.[7]

Chemical hyperalgesia (cold-allodynia/acetone drop test)

The cold allodynia was assessed by spraying a 100 μl of acetone onto the surface of the paw of the rat (placed over a wire mesh), without touching the skin. The response of the rat to acetone was noted for 20 s and was graded on a four-point scale as defined earlier.[8]

0. No response,

1. Quick withdrawal, flick or stamp of the paw,

2. Prolonged withdrawal or repeated flicking,

3. Repeated flicking of the paw with licking of the paw.

Acetone was applied thrice to the hind paw, with a gap of 5 min between the acetone applications and the individual scores noted in 20 s interval were added to obtain a single score over a cumulative period of 60 s. The minimum score was 0, while the maximum possible score was 9.

Mechanical dynamic allodynia (paint-brush test)

The “paint-brush” behavioral test is used to explore dynamic responses to a mechanical stimulus. The response to smooth paintbrush has been described as allodynia because normal rats never withdraw from this stimulus. It has been established that dynamic mechanical allodynia is mediated by peripheral low-threshold, large myelinated Aβ-fibers. The rat was placed in a cylinder with a wire mesh floor, and a smooth paint-brush was used to rub the plantar area of hind paw from the heel to the toes as a stimulus. The stimulus was applied 5 times with a 5 s interval, and the number of withdrawals was noted (between 0 and 5). The same procedure was repeated twice, after a gap of 5 min and the total number of withdrawals (in three tests) was added to obtain a single cumulative score of mechanical dynamic allodynia with a minimum value of 0 and maximum of 15.[9]

Collection of biological samples

At the end of treatment schedule (21 days) blood samples were withdrawn from the retro orbital venous plexus of rats without any coagulant for the separation of serum by centrifugation at 2500 rpm for 15 min and stored at 4°C until further analysis for serum total protein, serum nitrite and in vivo anti-oxidant parameters like super oxide dismutase (SOD), catalase, reduced glutathione and lipid peroxidation (LPO) using standard procedures.[101112131415] Then the rats were sacrificed by decapitation. The ligated sciatic nerve was excised, washed with normal saline and sliced into two pieces. One-half was homogenized for estimating myeloperoxidase (MPO) activity, calcium estimation and the other half was stored in 10% formalin and further used for histopathological studies.

Myeloperoxidase activity was measured using a procedure similar to that documented by Krawisz et al., and modified by Hillegass et al., Sciatic nerve tissues were homogenized in 50 mm potassium phosphate buffer (pH 6.0), and centrifuged at 2500 rpm (for 10 min); pellets were resuspended in 50 mm phosphate buffer containing 0.5% HETAB. After three freeze and thaw cycles, with sonication between cycles, the samples were centrifuged at 2500 rpm for 10 min. Aliquots (0.3 mL) were added to 2.3 mL of the reaction mixture containing 50 mM phosphate buffer, o-dianisidine, and 20 mmol H2O2 solution. The presence of MPO was measured at 460 nm for 3 min. MPO activity was expressed as U/g tissue. One unit of MPO activity was defined as that degrading 1 μ mol peroxide/min at 25°C.[16] Calcium was measured by colorimetric measurement with ortho-cresolphthalein complex.[17]

RESULTS

The ethanol soluble carbohydrate content of EEAV was found to be 100 mg/dL from phenol-sulfuric acid test.

Effect of ethanolic extract of A. vera on nociceptive threshold

Thermal sensitivity was assessed by PWL using both cold water and hot water immersion tests. In both the models of thermal sensitivity, SCNL caused a significant decrease in score of PWL in Group II when compared with Group I on day 7, 14 and 21. Treatment with gabapentin and variable doses (100 and 300 mg/kg, p.o.) of EEAV significantly increased the score of PWL in both the models of thermal sensitivity indicating increased threshold of pain as compared to disease control group (II) on day 7, 14 and 21 respectively [Tables 1 and 2].

Table 1

Effect of EEAV on paw withdrawal latency by cold water immersion

Table 2

Effect of EEAV on paw withdrawal latency using hot water immersion

In chemical and mechanical hyperalgesia models, disease control group showed a significant decrease in the PWL score when compared to normal control group on day 7, 14 and 21. Gabapentin and EEAV (100 and 300 mg/kg, p.o.) treated groups produced significant increase in score of PWL when compared to disease control group on day 7, 14 and 21 [Tables 3 and 4].

Table 3

Effect of EEAV on paw with drawal latency using acetone drop test

Table 4

Effect of EEAV on paw withdrawal latency using paint brush test

Effect of ethanolic extract of A. vera on biochemical parameters

Serum protein

There was a significant decrease in the serum total protein levels in Group II when compared to normal group (Group-I) which was significantly reversed with the treatment of gabapentin and EEAV at both the doses [Figure 1].

Figure 1

Effect of ethanolic extract of Aloe vera on serum protein and serum nitrite. Units: Serum protein – g/dL; serum nitrite – μg/mL; all values are shown as mean ± standard error of the mean and n = 8; *indicates P < 0.05 when compared with normal; **indicates P < 0.05 when compared with control group

Serum nitrite

The serum nitrite levels significantly increased in Group II when compared to normal control (Group I). The EEAV treatment (100 and 300 mg/kg, p.o.) significantly decreased the levels of serum nitrite when compared to disease control animals. Similar decrease in serum nitrite levels was also observed in gabapentin treated animals [Figure 2].

Figure 2

Effect of ethanolic extract of Aloe vera on calcium and myeloperoxidase. Units of calcium (ppm/mg); myeloperoxidase (U/g); all values are shown as mean ± standard error of the mean and n = 8; *indicates P < 0.05 when compared with normal; **indicates P < 0.05 when compared with control group

Calcium and MPO levels in sciatic nerve homogenate: There was a significant increase in calcium and MPO levels in disease control group (II) when compared to the normal group (I). Gabapentin treated group (III) and groups treated with 100 and 300 mg/kg, p.o. of EEAV respectively showed significant decreased levels of calcium and MPO on comparison with control [Figure 2].

Effect of ethanolic extract of A. vera on serum in vivo anti-oxidant parameters

After SCNL, on day 21 there was a significant decrease in the levels of in vivo anti-oxidants like SOD, catalase, reduced glutathione and significant increase in malondialdehyde levels in Group II on comparison with Group I supporting the role of oxidative stress in neuropathic pain. Treatment with gabapentin and EEAV (100 and 300 mg/kg, p.o.) significantly increased the levels of in vivo anti-oxidant like SOD, catalase, reduced glutathione and significantly lowered the malondialdehyde levels [Figures 3 and 4].

Figure 3

Effect of ethanolic extract of Aloe vera on super oxide dismutase (SOD), catalase and glutathione (GSH). Units of SOD: U/g; catalase: H2O2 consumed/min/mg of tissue; GSH: μg of GSH/g of tissue; all values are shown as mean ± standard error of the mean and n = 8; *indicates P < 0.05 when compared with normal; **indicates P < 0.05 when compared with control group

Figure 4

Effect of ethanolic extract of Aloe vera on lipid peroxidation (LPO). Units of LPO (nmoles of malondialdehyde/mg of tissue); all values are shown as mean ± standard error of the mean and n = 8; *indicates P < 0.05 when compared with normal; **indicates P < 0.05 when compared with control group

Effect of ethanolic extract of A. vera on histopathology of the sciatic nerve

A significant damage denoted by widened endoneural spaces disrupted myelin sheath, and swollen Schwann cells was observed in the photomicrographs of sciatic nerve section of Group II animals upon SCNL when compared to the Group I animals. Treatment with gabapentin has showed restoration of damage caused by SCNL comparable to the normal animals. EEAV also partially restored the damaged sciatic nerve supporting the efficacy of EEAV against neuropathic pain [Figure 5].

Figure 5

Photo micrographs of the sciatic nerve stained with toulidine blue (×40). (a) Group I (Normal) = Treated with vehicle; (b) Group II (Control) = Sciatic nerve ligation (SCNL); (c) Group III (Standard) = SCNL + Gabapentin (9 mg/kg, p.o.); (d) Group IV (Low dose) = SCNL + Aloe vera extract (100 mg/kg, p.o.); (e) Group V (High dose) = SCNL + A. vera extract (300 mg/kg, p.o.)

DISCUSSION

Neuropathic pain can be very severe and disabling, with significant functional, psychological, and social consequences. Although precise estimates of the prevalence of neuropathic pain are not available, it is more common than that has generally been reported. Evidence-based treatment recommendations which take into account clinical effectiveness, adverse effects, influence on the quality of life and cost for the pharmacologic management of chronic neuropathic pain are necessary.[18]

Animal studies suggest a gender difference in pain processing, but little is known about influence of estrogens on drug management. Sex differences in pain perception exist due to its differential modulation by estrogens and androgens with females typically reporting higher sensitivity to noxious stimuli and a higher incidence of various painful conditions.[19] Hence female rats were used in the present study.

Sciatic nerve ligation resembles human neuropathy resulting from trauma of peripheral nerves, with some functional preservation of innervations (nerve entrapment or compression). This model is one of the most commonly used models because it is reliable and easily reproducible.[20] Gabapentin is considered a drug of choice for treating neuropathic pain. Hence, gabapentin was used as a standard to compare the protective effect of EEAV in the present experimental model of neuropathic pain.

Many works with aloe gel or aloe aqueous extract for different therapeutic purposes were reported. But, the purpose of selecting ethanolic extract in the present study was to extract acemennan, a water insoluble polysaccharide claimed to be a potent polysaccharide with anti-inflammatory activity. The amount of acemennan obtained in the EEAV was found to be 100 mg/dL from phenol-sulfuric acid test.

Peripheral nerve injury produces a persistent neuropathic pain characterized by spontaneous pain, allodynia and hyperalgesia. SCNL was reported to induce an ipsilateral cold allodynia, thermal hyperalgesia and oxidative damage in the sciatic nerve. Sensitization of primary afferent nerves has been suggested as one of the mechanisms involved in hyperalgesic action.

Similar thermal hyperalgesia, cold and mechanical allodynia was observed in animals that were significant after day 7, post-SCNL. EEAV improved the pain threshold and attenuated thermal hyperalgesia, cold and mechanical allodynia in a dose-dependent manner suggesting its therapeutic potential against neuropathic states.

Aloe anthraquinones suppress cytolytic T-lymphocytes in favor of suppressor cells. Furthermore, anthraquinones decrease cytokine production and interleukin-2 (IL-2) mRNA expression in activated T lymphocytes, thereby decreasing chemotaxis. More recent studies have demonstrated that the anthraquinone emodin decreases plasma levels of the cytokines IL-2 and tumor necrosis factor alpha (TNF-α), while increasing IL-10 (which itself down-regulates IL-2 and TNF-α cytokine activity).[21] Acemennan has been reported to inhibit chemotaxis of polymorphonuclear leukocytes or have inhibit eicosanoid, histamine and bradykinin formation.[2223] From the protective activity shown by EEAV in the present study, it can be proposed that the presence of active constituents like acemennan and anthraquinones might be responsible for increasing threshold against pain transmission.

Sciatic nerve ligation shows significant oxidative damage in the sciatic nerve as indicated by rise in LPO and nitrite concentration as well as depletion of reduced glutathione and catalase activity. Reactive oxidant species are critically involved in the development and maintenance of neuropathic pain, while administration of nontoxic doses of free radical scavengers could be useful for the treatment of neuropathic pain. Increasing evidences suggests a key role of oxidative stress in neuropathic pain and other neurological diseases, but the precise mechanisms that underlie pain and oxidative stress remains unclear. Malmberg and Basbaum suggest that endoneural LPO increases as a consequence of chronic constriction injury in the sciatic nerve.[24]

Treatment with EEAV significantly reduced oxidative damage by attenuating the increased lipid peroxides, nitrite concentration and restoration of depleted anti-oxidant enzymes. It seems that the beneficial effects of A. vera could be because of its anti-oxidant property.

A significant decrease in serum nitrite levels was observed on treatment with EEAV, which supports the conclusion that A. vera may also be acting as a NOS inhibitor, thus proving effective against pain induced by SCNL. The protection of EEAV on sciatic nerve was also confirmed from the histopathological studies supporting the other results, confirming the therapeutic potential of EEAV for treating neuropathic pain.

CONCLUSION

The present study proposes that anti-oxidant property of EEAV against reactive oxygen species, decreased chemotaxis of neutrophils as shown by decreased MPO levels may be responsible for the protective effect of A. vera against neuropathic pain. Further exploration of reasons for decreased calcium content in the sciatic nerve will elucidate the probable mechanism for the analgesic effect of EEAV against neuropathic pain. Doing so will help in developing EEAV as complementary alternative for the management of neuropathic pain.