For the present study, Divya-Peedantak-Kwath (DPK) (batch no: A-PTH037, manufactured date: July 2019) was obtained from Divya Pharmacy, A-1 Industrial Area, Haridwar, India. Culture media RPMI-1640, fetal bovine serum, and antibiotic/antimycotic mixture were obtained from Gibco, and cytokines interleukin IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) ELISA kits were purchased from BD Biosciences. Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, MO, United States). Phorbol 12-myristate 13-acetate (PMA) was purchased from Fisher Scientific, United States. Paclitaxel injection IP (brand name—MITOTAX 100; marketed by Dr Reddy’s Laboratories Ltd., Hyderabad, India) was procured from local pharmacy; gabapentin was procured from TCI Chemical Industry Co. Ltd., Tokyo, Japan.

Chemical composition of DPK was analyzed through high-performance liquid chromatography (HPLC) (Waters Corporation, United States); instrument equipped with a binary pump (1525), PDAD (2998) and an autosampler (2707). Separation was achieved using a Shodex C18-4E (5 μm, 4.6 * 250 mm) column through binary gradient elution. The two solvents, designated as A and B, were used for the analysis. Both A and B had 0.1% orthophosphoric acid. But in case of B, it was in a mixture of acetonitrile and water in 88:12 ratios, while it was in water for A. pH of both solvents was set to 2.5 with diethylamine. The gradient program for the solvent system used was 95–85% A for 0–10 min, 85% A for 10–15 min, 85–80% A for 15–20 min, 80–70% A for 20–30 min, 70–40% A for 30–40 min, 40–10% A for 40–45 min, 10–95% A for 45–46 min, and 95% A for 46–50 min, with a flow rate of 1.0 ml/min 1.0 g of the sample was suspended in 20 ml methanol: water (10:90) mixture sonicated for 30 min, centrifuged at 10,000 rpm for 5 min, and filtered through a 0.45-µm nylon filter before loading on to the column. Then, 10 µl of the test solution was used for analysis. The column temperature was maintained at 35°C. Wavelengths were set to 270 nm (for gallic acid, vanillic acid, corilagin, rutin, and ellagic acid) and 325 nm (for neochlorogenic acid and cryptochlorogenic acid). Standard compounds were purchased from Natural Remedies Pvt. Ltd, Sigma Aldrich, Bangalore, and ChemFaces, China, and were dissolved in methanol at recommended concentrations.

THP-1 cell line was obtained from the ATCC-licensed cell repository National Centre for Cell Science, Pune, India. These cells were cultured in RPMI-1640 media; supplemented with 10% heat-inactivated fetal bovine serum, in the presence of penicillin–streptomycin (100 U/ml), sodium pyruvate (1 mM), and l-glutamine (4 mM); and grown at 37°C in a CO₂ incubator with 5% CO₂. All experiments were done with cells between passage numbers 8 and 10.

Water-extracted DPK powder was dissolved in incomplete RPMI-1640 culture medium. THP-1 cells were plated in a 96-well plate at a density of 10,000 cells per well and differentiated to macrophages with 20 ng/ml PMA. Following a 24-h exposure, the differentiation medium was replaced with fresh RPMI 1640, and cells were allowed to rest for 24 h. Then, 500 ng/ml of LPS was used to induce inflammation in these macrophages. The cells were treated for 24 h with different concentrations of DPK (1, 10, 100, 300, and 1,000 μg/ml) diluted in media. Cells were pretreated with DPK extract for 24 h before inducing inflammation with LPS. Cell supernatants were collected in a 96-well plate for measuring the levels of pro-inflammatory cytokines: TNF-α, IL-6, and IL-1β. Cytokine levels were measured using ELISA kits (BD Biosciences), according to the manufacturer’s protocol. Absorbance was recorded at 450 nm using an EnVision microplate reader (Perkin Elmer, United States). After washing the cells with PBS, 100 µl alamarBlue (15 μg/ml) was added to each well and incubated for 3 h at 37°C. Subsequently, fluorescence was measured using the EnVision microplate reader (Perkin Elmer, United States) at 530 (excitation) and 590 (emission) nm, and cell viability percentage was calculated.

Six- to 8-week-old male CD-1 mice weighing 20–25 g were procured from Charles River Laboratory–licensed supplier, Hylasco Biotechnology Pvt. Ltd., Hyderabad, India. All animals were housed in polypropylene cages at a controlled temperature of 22 ± 3°C with a relative humidity of 60–70% under 12:12-h light and dark cycles in a registered animal house (registration number: 1964/PO/RC/S/17/CPCSEA). The animals were supplied with standard pellet diet (Purina Lab Diet, St. Louis, MO, United States) and sterile filtered water ad libitum. The study protocol was approved by the Institutional Animal Ethical Committee of Patanjali Research Institute vide IAEC approval number PRIAS/LAF/IAEC-069. All the experiments were performed as per the relevant guidelines and regulations.

The animal equivalent doses of DPK for mice study was estimated based on the body surface area. The human recommended therapeutic dose of the DPK is 5 g powder boiled in 400 ml of water until ∼100 ml decoction remains, twice a day. Accordingly, total human dose is 10 g/70 kg/day. For DPK sample preparation, 500 g of DPK was boiled in 20 L distilled water and was reduced to 1.8 L of decoction, which was concentrated, filtered, and dried to yield 58.1 g of dry powder. Based on a decoction yield of 11.6%, the calculated human dose is 1.16 g/70 kg/day (16.6 mg/kg/day). Animal equivalent doses (mg/kg) for mouse were calculated by multiplying human equivalent dose (HED) (mg/kg) by a factor of 12.3 (Nair and Jacob, 2016). Resultant therapeutic equivalent doses for mouse were found to be 204 mg/kg. Therefore, 205 mg/kg (round off) has been taken as mouse mid-doses (HED). The low dose (69 mg/kg body weight) is one-third of the HED, and the high dose (615 mg/kg body weight) is three times the HED for mice.

The CIPN animal model was generated as previously described (Thangamani et al., 2013; Kaur and Muthuraman, 2019). Eight- to 10-week old male CD-1 mice were randomized based on body weights and divided into six different groups consisting of six animals each. The details of randomization, dosage, and associated descriptions are depicted in Table 1. Due to one mortality observed in G3 (GABA), animal sample size was revised to N = 5. Animals of the group G1 served as normal control. PTX was administered intraperitoneally (i.p.) at 2 mg/kg body weight for six consecutive days to those of groups G2 to G6. PTX-treated animals were assessed for pain sensitivity. Animals that developed neuropathic pain were selected for the experiment and randomized into different treatment groups (G2–G6). Animals of group G2 served as disease control (DC) and were treated with 0.25% Na-CMC. The reference drug gabapentin (GABA) was administered intraperitoneally to the animals of group G3 (GABA) at 100 mg/kg body weight. Animals of groups G4 (DPK-69), G5 (DPK-205), and G6 (DPK-615) were treated orally with Divya-Peedantak-Kwath (DPK) at 69, 205, and 615 mg/kg body weight, respectively. All drug treatments were given for two weeks. The neurobehavioral monitoring of the animals through hot plate, tail flick latency, Von Frey, and Randall–Selitto tests was performed on different days, before and after PTX treatment. Body weights of the animals were measured every day for fifteen days (day 1–day 15) from the day when PTX administration was started. The change in body weight of each animal was calculated before and after the development of CIPN and after subsequent treatment. Animal feed and water consumption were recorded daily for the whole study duration. TABLE 1

Group identification, dosage, and associated description used in in vivo study.

In order to establish the CIPN model, paclitaxel was administered intraperitoneally daily for 6 days. These 6 days are denoted as day 0–day 6. All the basal pain behaviors were assessed on day 0. Subsequently, hot plate assay was carried out on day 6, day 11, and day 16; tail flick and Von Frey assays on day 7, day 12, and day 17; and Randall–Selitto on day 9, day 13, and day 19.

Sciatic nerves were harvested from humanely sacrificed animals at the end of the experiment and stored in neutral buffered formalin for further processing for H&E staining according to procedure described earlier (Balkrishna et al., 2019a; Balkrishna et al., 2019b). The slides were examined under a microscope by a veterinary pathologist for histopathological lesions. The severity of the observed lesions was scored as 1 = minimal (<1%), 2 = mild (1–25%), 3 = moderate (26–50%), 4 = marked (51–75%), and 5 = severe (76–100%). The distribution of the lesions was recorded as focal, multifocal, and diffuse. Images of the histological slides (H&E) were captured at low (×10) and high (×40) magnifications using an Olympus Magnus microscope camera and processed by Olympus MagVision image analysis software.

The levels of reduced (GSH) and oxidized (GSSG) glutathione were measured according to Hissin and Hilf (1976). The isolated sciatic nerve tissue samples were homogenized in 0.1 M phosphate buffer (pH 8) containing 25% metaphosphoric acid. The homogenates were clarified through centrifugation at 10,000 g for 20 min at 4°C. Reduced glutathione (GSH) content was determined by incubating clarified homogenate with o-phthaldialdehyde (OPT) at room temperature for 15 min in dark. Fluorescence was measured at 350 (excitation) and 420 (emission) nm using the EnVision microplate reader (Perkin Elmer, United States). Levels of oxidized glutathione (GSSG) were determined by first incubating the clarified tissue homogenates with N-ethylmaleimide (0.04 M) for 30 min in dark and, subsequently, with 0.1 N NaOH and OPT solution for another 15 min. Fluorescence was measured at 350 (excitation) and 420 (emission) nm as mentioned earlier. Lipid peroxidation was monitored through the levels of malodialdehyde (MDA) in the clarified tissue homogenates according to Heath and Packer (1968). This assay is based on the reaction of MDA with thiobarbituric acid (TBA) forming an MDA–TBA adduct that absorbs at 532 nm. Clarified tissue homogenates were incubated with three volumes of 0.5% TBA (diluted in 0.1 M phosphate buffer, pH 8) at 95°C in a water bath for 25 min. The reaction was stopped by incubating the samples on ice. Absorbance was measured at 532 and 600 nm using the EnVision microplate reader (Perkin Elmer, United States). Absorbance values measured at 600 nm were subtracted from MDA–TBA complex values at 532 nm. MDA concentration was calculated using the Lambert–Beer equation with an excitation coefficient ε ^M = 155 mM⁻¹cm⁻¹.

TNF-α levels in the serum were estimated using an ELISA kit (BioLegend, Inc., San Diego, CA, United States) according to the manufacturer’s instructions. The plate was read at 450 nm using the EnVision microplate reader (Perkin Elmer, United States).

Experiments were performed in triplicates and quantitative data represented as mean ± SEM. Two groups were compared by an unpaired t-test. For comparing three or more groups, one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test was used. For comparisons between three or more groups for different time points, two-way ANOVA followed by Tukey’s multiple comparisons test was used. Observations were considered statistically significant at p-value ≤ 0.05.

DPK is a herbal decoction consisting of 28 medicinally important plants (Table 2). Naturally, it is expected to be enriched with several active compounds. In fact, the herbs present in DPK are meticulously chosen to target the causative elements of CIPN, namely, oxidative stress, inflammation, and subsequent immune reactions. Therefore, it was imperative to check the active ingredients present in DPK that can target oxidative stress, reduce inflammation, and modulate immunological reactions, besides subduing the nociception and pain associated with CIPN. Employing the high-performance liquid chromatographic (HPLC) technique, the major compounds identified in DPK are gallic acid (1.009 μg/mg) and neochlorogenic acid (1.067 μg/mg). Besides, cryptochlorogenic acid (0.804 μg/mg), ellagic acid (0.243 μg/mg), corilagin (0.124 μg/mg), vanillic acid (0.078 μg/mg), and rutin (0.069 μg/mg) were also detected (Figures 1A,B; Table 3).

Given the fact that DPK is practically a storehouse of anti-inflammatory agents, we evaluated its efficiency in attenuating inflammation on differentiated THP-1 cells in vitro. PMA-differentiated, LPS-inflamed human THP-1 cells were treated with different concentrations (1, 10, 100, 300, and 1,000 μg/mg) of DPK. LPS-treated and -untreated differentiated THP-1 cells, respectively, serving as disease (0) and normal (NC) controls did not receive any DPK treatment. Pro-inflammatory cytokines, namely, interleukin-6 (IL-6), interleukin 1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) have been implicated in neuroinflammation and neuropathic pain (Leung and Cahill, 2010; del Rey et al., 2012; Hung et al., 2017). Therefore, we monitored the effect of DPK treatment on these cytokines released in LPS-induced THP-1 cells. The levels of IL-6, IL-1β, and TNF-α were, respectively, 3, 2.8, and 5.6 times higher in disease control (0) cells with inflammation (Figures 2A–C). Noticeable reductions in IL-6 levels from 353.9 (±4.7) pg/ml in (0) group to 242.2 (±8.8) pg/ml and 74.1 (±13.1) pg/ml were observed when the cells were treated with DPK at 300 and 1,000 μg/mg concentrations, respectively (Figure 2A). Effects on IL-1β levels were visible with DPK treatments at 100 μg/mg or more. It reduced from 540.83 (±22.8) pg/ml in (0) group to 344 (±28.4), 320 (±2.3), and 293 (±27.6) pg/ml, when treated with 100, 300, and 1,000 μg/ml of DPK, respectively (Figure 2B). Significant reduction in the level of TNF-α relative to the disease control (0) cells (3,685.0 ± 33.3 pg/ml) was observed with 10 μg/mg DPK treatment (3,070.1 ± 36.9 pg/ml) onward. However, we did not observe dose dependency in the reduction in TNF-α (Figure 2C). Taken together, these observations showed that DPK possess strong anti-inflammatory effects. In order to ensure that PMA, LPS, DPK, or their cotreatments did not have any untoward effects on the cells and that the model was suitable for evaluation of inflammation, cell viability of THP-1 cells was checked through alamarBlue assay, conforming to the described experimental regimen. Cells were equally viable in the control and experimental groups, confirming the cytosafety of DPK in the treatment regimen and also lending the credibility to the experimental setup (Figure 2D).

The mouse model of CIPN was generated through intraperitoneal injection of PTX at a human-relevant clinical dose. The experimental design and regimen have been explained in detail in the Material and Methods (Chemotherapy-Induced Peripheral Neuropathy Animal Models and Treatment Groups), Table 1, and schematically represented in Figure 3A. The effect of DPK on general health and metabolism was monitored through change in the body weights of the animals and alteration in their food and water intake habits. The PTX-induced disease control (DC) group showed approximately 9% increase in body weight by day 14, whereas that of normal control (NC) group was 3% by the same day. The DC group showed a statistically significant more increase in body weight than the normal control (NC) animals. Interestingly, an increase in body weights was under control by day 13 and day 14 in the DPK-615 group, receiving a daily oral dose of 615 mg/kg DPK. The average body weights in this group were significantly lower when than those of the DC and NC groups. However, similar effects were not observed in DPK-69 and DPK-205 groups, which received daily oral doses of 69 and 205 mg/kg of DPK, respectively. This effect was also not observed in the group receiving gabapentin, the FDA-approved drug for treating CIPN (Figure 3B). Food and water intake were similar among the animals across different groups (data not shown). This suggested that observed increase in the body weight of the DC group is not due to any change in the eating and drinking habits of the animals, rather most likely due to some underlying metabolic alterations experienced while establishing the CIPN mouse model.

PTX has been shown to induce heat hyperalgesia, mechano-hyperalgesia, mechano-allodynia, and allodynia (Scripture et al., 2006). Therefore, the pharmacological effects of DPK on PTX-induced neuropathic pain sensitivity was analyzed using the hot plate test, tail prick latency test, Von Frey test, and Randall–Selitto test (Figures 4A–D). Efficacy of DPK in assuaging heat hyperalgesia was monitored through the hot plate test (Figure 4A). PTX administration reduced the latency times to almost half (from average 12.29 ± 0.24 s to around 7.78 ± 0.18 s) by day 6 in all the groups, except NC, one that did not receive the chemotherapeutic treatment. A significant increase in the latency times of the groups receiving treatments for CIPN was observed over time. Latency time of the group receiving gabapentin increased from an average 7.58 ± 0.80 to 9.03 ± 0.45 s by day 11, a significant improvement over the DC group, in which the latency time further reduced to around 5.29 (±0.20) s over this period. An increase in latency time (8.52 ± 0.88 to 9.24 ± 0.75 s) in the group receiving DPK at 206 mg/kg body weight was comparable to the GABA group at day 11. However, the group receiving DPK at 69 mg/kg body weight showed only a marginal increase (7.76 ± 0.39 to 8.22 ± 0.33 s) in latency time over this period. The most significant increase in latency time, from 7.52 (±0.88) to 10.28 (±0.91) s, was observed in the group that received DPK at 615 mg/kg body weight. This increase was even more pronounced than that observed in the GABA group. Moreover, the increase in latency times of all the treatment groups was significant when compared to the DC group. The improvement was more noticeable by day 16 which witnessed a sharp increase in latency times 11.76 (±0.49) and 10.92 (±0.36) s, respectively, for DPK-615 and DPK-205 groups. The GABA group showed a trend similar to the DPK-205 group, with its average latency time increasing to 11.08 (±0.53) s. However, DPK-69, with its average latency time increasing to only 8.43 (±0.3) s, did not show a sharp increase in latency time as other DPK groups, although it was statistically significant (p < 0.01) relative to the DC group. The increase in latency times observed in all other treatment groups was significantly more when compared to DC.

The tail flick latency test was conducted to evaluate the modulation of nociception and relieving of mechano-hyperalgesia by DPK treatment (Figure 4B). A threshold around 8 (±0.18) s of latency was established which was maintained in the NC group. Induction of CIPN with PTX reduced this threshold significantly, to an average 3.65 (±0.12) s in the treatment groups by day 7. In the DC group, this was even lower (1.89 ± 0.17 s). This reduction of latency threshold upon CIPN induction reflects allodynia. Treatment with gabapentin (GABA group) increased the average latency time to 5.69 ± 0.09 s by day 12 and 7.15 ± 0.09 s by day 17. Latency time in the DPK-615 group was around 3.6 ± 0.11 s by day 12 but caught up with the GABA group at 7.13 ± 0.08 s by day 17. These values were noticeably higher than those of the DC group. The increase in latency times in DPK-69 and DPK-205 trailed behind. DPK-205 witnessed an increase in latency time to 4.53 ± 0.15 s by day 12 and 6.14 ± 0.09 s by day 17. Latency times for DPK-60 after day 12 and day 17 were around 3.79 (±0.17) and 5.16 (±0.13) s, respectively. While all the treatment groups showed increases in latency times reflecting amelioration of the inflicted allodynia, latency time of the DC group remained significantly lower than that of the treatment groups. In fact, it reduced to 1.70 (±0.05) s by day 12 from 1.89 (±0.17) s on day 7 and further to 1.58 (±0.08) s by day 17, although the reduction was not visibly discernible.

The effect of DPK treatment in ameliorating mechano-allodynia due to PTX-induced peripheral neuropathy was evaluated by using the Von Frey test (Figure 4C). The average paw withdrawal threshold (PWT) for all the groups was around 7.72 ± 0.08 g before PTX treatment. PWT reduced to average 3.49 ± 0.03 g by day 7 in the treatment groups once the PTX dosing was initiated. For this DC group, this was even lower at 2.99 ± 0.15 g. These were significant reductions across the experimental groups when compared to the PWT of the NC group that continued to be around 8.43 ± 0.15 g till day 12, showing a marginal increase by day 17 (8.64 ± 0.09 g). PWT increased in the GABA group to 5.49 ± 0.12 g by day 12 and 7.25 ± 0.04 g by day 17. The increase in PWT in the DPK-615 group also followed a trend similar to that of the GABA group (5.21 ± 0.26 g by day 12 and 7.53 ± 0.16 g by day 17). The PWT increase in DPK-205 was similar to that of GABA and DPK-615 groups till day 12 (5.12 ± 0.26 g), beyond which it fell behind to 6.24 ± 0.13 g by day 17. A discernible increase in PWT in the DPK-69 group was observed only by day 17 (5.08 ± 0.14 by day 17 vs. 3.47 ± 0.05 g of day 7). However, PWTs of all these groups were significantly more than those of the DC group, in which the threshold kept decreasing, albeit, marginally.

Allodynia or increased pain sensation in PTX-induced neuropathy can be due to neuroinflammation (Myers et al., 2006; Ji et al., 2018). Therefore, the Randall–Selitto test was used to assess the effect of DPK treatment on modulation of this pain sensitivity (Figure 4D). While the normal PWT in all the groups was observed to be 21.2 ± 0.18 g, it reduced to around 5.5–9 g after PTX dosing by day 9. Following gabapentin treatment, PWT increased to 11.69 ± 0.51 and 17.54 ± 0.31 g by day 13 and day 19, respectively. A visible increase in PWT in case of DPK-69, DPK-205, and DPK-615 groups was observed to be 8.93 ± 0.22, 13.86 ± 1.17, and 16.31 ± 0.45 g, respectively, only by day 19. Meanwhile, PWT in the DC group continued to decrease further to 3.93 ± 0.23 and 3.63 ± 0.22 g by day 13 and day 19, respectively. All of these observations demonstrated that DPK can be a very potent modulator of PTX-induced pain sensitivity.

The effect of DPK treatment on PTX-induced oxidative stress in peripheral nerve tissue was evaluated ex vivo by monitoring tissue levels of MDA, GSSG, and GSH:GSSG (Figures 5A–C). The DC group is expected to experience enhanced lipid peroxidation due to PTX treatment which was reflected as a significantly increased MDA level (p < 0.01) when compared to the NC group. Gabapentin and DPK treatments significantly (p < 0.01) reduced the MDA levels in the respective treatment groups (Figure 5A). As expected, the GSSG level in the DC group was more than double of that observed in the NC group (117.34 ± 10.61 vs. 51.45 ± 7.71 μM/mg of tissue, p < 0.05). Treatment with gabapentin although showed visible reduction in the GSSG level, the change was not statistically significant when compared to that of the DC group. However, GSSG levels in all the DPK-treated groups were significantly lower (p < 0.01) relative to DC groups (Figure 5B). Likewise, the GSH:GSSG in all the treatment groups were noticeably high when compared to the DC group. The differences were statistically significant (p < 0.05 for GABA and DPK-205 groups and p < 0.01 for the DPK-615 group). GSH:GSSG for the DPK-69 group was visibly more than that of the DC group, but the difference was not statistically significant (Figure 5C). We also checked the effect of DPK treatment on the serum levels of pro-inflammatory cytokine, TNF-α in study animals (Figure 5D). The mean serum TNF-α was significantly raised in the DC group when compared to the NC group (20.25 ± 1.36 pg/ml vs. 13.50 ± 0.83 pg/ml, p < 0.01). Treatment with gabapentin reduced the serum TNF-α level to 5.72 ± 0.84 pg/ml (DC vs. GABA, p < 0.001). Treatments with DPK showed a dose-dependent reduction of serum TNF-α, and the changes were all statistically significant when compared to the DC group [20.25 ± 1.36 pg/ml in DC vs. 15.94 ± 1.36 (p < 0.05), 12.50 ± 0.34 (p < 0.001), and 10.23 ± 1.93 (p < 0.001) pg/ml in DPK-69, DPK-205, and DPK-615 groups, respectively]. Taken together, these observations indicate that DPK treatment alleviates the oxidative stress and concomitant inflammation imposed by PTX treatment.

PTX causes axonal degeneration, swelling, and lymphocytic infiltration (Peters et al., 2007; Tasnim et al., 2016). Thus, efficacy of DPK in treating these conditions was evaluated through histological staining of sciatic nerve tissue and subsequent microscopic analyses (Figure 6). These pathological features have been scored and quantitatively represented in Figures 7A–D. Representative micrograph from the NC group shows intact axons (A), Schwann cells (SC), and myelin sheath (MS) (Figure 6A). Consequently, no lesion score showed up for this group (Figures 7A–D). PTX treatment shows degenerated and swollen axons, indicated as AD and AS, respectively, in the micrograph. The image also shows infiltrated lymphocytes (L) (Figure 6B). Treatments with gabapentin and DPK (at 205 and 619 mg/kg body weight doses) showed visible reduction in these pathological features in the respective images (Figures 6C,E,F). However, the group receiving DPK at 69 mg/kg body weight did not show much improvement with respect to these features (Figure 6D). Individual lesion scores for axonal degeneration, swelling, and lymphocytic infiltration as well as the total one for the DC group were obviously the maximum. The lesion score for axonal degeneration although reduced visibly in GABA group, the change was not statistically significant. Similarly, reductions in the lesion score of axonal degeneration observed in DPK-treated groups were also not statistically significant. The changes in DPK-205 and DPK-619 groups were comparable to those in the GABA group (Figure 7A). Lesion scores for axonal swelling in both GABA and DPK-619 groups were significantly lower (p < 0.05) when compared to the DC group. Although, the lesion score for axonal swelling in the DPK-205 group was comparable to GABA and DPK-619 groups, it did not show statistical significance when compared to the DC group. Lesion score for axonal swelling in the DPK-69 group did not show much reduction and was comparable to the DC group (Figure 7B). Lesion scores for infiltrating lymphocytes for each group exhibited a pattern similar to that shown by the lesion score for axonal swelling, except that the reduction in infiltrating lymphocytes in case of GABA and DPK-619 groups were more pronounced (p < 0.01) when compared to the DC group (Figure 7C). In the total lesion scoring, in addition to the GABA and DPK-619 groups, the DPK-205 group also showed statistically significant reduction relative to the DC group. However, the reduction in the latter was less pronounced than the former ones (p < 0.05 in case of DPK-205 vs. p < 0.01 in cases of GABA and DPK-619). Total lesion score for DPK-69 did not show statistically significant reduction when compared to the DC group, rather total lesion scores for both appeared comparable (Figure 7D). Altogether these observations showed that DPK treatment, albeit at higher doses, can assuage the etiology of PTX-induced neuropathy. Considering that DPK is an herbal decoction, higher dose may not be an issue with respect to toxicity. In fact, the recommended doses of gabapentin have reported side effects which may be not the case for DPK.