Evidence of Tumor Suppression Activity of Withania Somniferous - Dissertation Example

?INTRODUCTION Since its discovery, naturally-derived compounds such as those from plants are preferred over synthetic drugs because it causes considerably less side effects such as nausea, anorexia, dizziness, headache, fever, impaired liver function, pleuropericarditis, kidney efficiency, and pulmonary disease (Pawar et al., 2011). In addition, it is much more easier to purify active compounds and elucidate its structure rather than make a synthetic compound before determining whether it is active against the disease or not. As more natural products are tested for their biological activity, more have been discovered for their various action against inflammatory diseases, microorganisms, or reproductive problems, to name a few. At present, plant products have already been used as a standard chemotherapeutic agent. For example, vinblastine (from periwinkle (Catharanthus)) and paclitaxel (from Yew tree (genus Taxus)) are already a part of chemotherapeutic options used against cancer (Choudhary et al., 2010). Because a high number of individuals still suffer from cancer, the search is still ongoing for the compound, both natural and synthetic, that can hopefully decrease the cancer-caused mortality rate considerably. Withania somnifera, commonly called as Ashwagandha, is a member of Solanaceae family, together with Nicotiana (tobacco), Solanum (potato), and Capsicum (pepper). In herbal medicine, W. somnifera has already been used against various health conditions. In Pakistan, it has been used against respiratory problems (Choudhary et al., 2010). In India, it has been recognized as an aphrodisiac and invigorating medicine (Choudhary et al., 2010). The plant is also used against intestinal ulcers, rectal bleeding and irritable bowel (Pawar et al., 2011). Several withanolides isolated from Ashwagandha were also found to possess anti-glycation, possibly against diabetes, and anti-pyretic effects (Choudhary et al., 2010). This study aimed to conduct a literature review on the studies that perform experiments on the tumor suppression activity of Withania somnifera. For each of the experiments featured in this paper, details about the methods, such as the method of extraction, the test subjects, and the statistical analyses, as well as the results are elaborated. EVIDENCE OF TUMOR SUPPRESSION ACTIVITY OF Withania somniferous In this literature review, four journal articles, ranging from 2003 to 2011, were looked into. Most of the methods used by these studies are in vitro, and only one using in vivo, although in an experimental model (rats). Google scholar was used as a search engine, with the results limited to 2000 to 2011. The search terms used were “Withania somnifera cancer”. Later in this paper, the quality of the research would be assessed mostly based on the methods used in the purification and activity assays. Activity of crude methanolic extract against NCI-H460 Compounds from the leaves and stems of W. somnifera were tested for its anti-proliferative activity against human lung cancer cell line NCI-H460 in vitro. In this study by Choudhary et al. (2010), tested for growth inhibitory and cytotoxic activities were the (1) crude methanolic extract of W. somnifera, (2-4) three isolates, and the positive control (5) doxorubicin. The isolates were obtained using silica gel chromatography. They were then characterized using mass spectrometry and NMR. One of the three compounds were identified as withaferin A, while the other two were found to be its chlorinated steroidal lactone and epoxide derivatives, respectively. In testing their activity, GI50, or the concentration causing 50% growth inhibition of NCI-H460 cells, and LC50, or the concentration causing the death of 50% of the same cancer cell line, were measured for all the five test substances. The obtained data were compared using one-way ANOVA and Duncan’s multiple range test (p < 0.05) using SPSS 17 program. Finally, the report also mentioned the presence of other studies that confirm the growth inhibitory activity of withaferin A against other cancer cell types such as pancreatic, ovarian, breast, colon, and prostate. Based on their reported GI50, none of them are as inhibited by withaferin A as NCI-H460 is. Anticarcinogenic activity of W. somnifera withanolides In the study by Jayaprakasam et al. (2003), a more extensive study was conducted. The anticarcinogenic activity of specific compounds isolated from Withania somnifera were tested on various cancer cell lines. Compounds were isolated from W. somnifera leaves using silica column chromatography with CHCl3 and MeOH step gradients. The peaked fractions underwent another chromatography separation using medium pressure liquid chromatography (MPLC), high pressure liquid chromatography (HPLC) and preparative thin layer chromatography (PTLC). The separation procedures resulted to 12 withanolide compounds, one of which, withaferin A, was used to make diacetylwithaferin A from pyridine and acetic anhydride. The isolated withanolides and diacetylwithaferin A were tested for their cytotoxic and growth inhibitory activities through 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay on various cancer cell lines. Adriamycin, or doxorubicin, and DMSO were used as postivie and negative control, respectively. NCI-H460 (lung), HCT-116 (colon), SF-268 (central nervous system), and MCF-7 (breast), were maintained using RPMI-1640 media with fetal bovine serum (FBS) from Gibco BRL. Doses used ranged from 0.088 ?g/ml. The absorbance of the resulting cell culture mixture was then measured at 575 nm. The study used multiple regression to produce lest-squares polynomial equations that describe the effects of withanolide exposure and concentration on cancer cell growth and viability. In turn, these equations were used to determine the minimum concentration that causes a 50% decrease in the cell count for each cell line. W. somnifera L-asparaginase Another study, conducted by Oza et al. (2010), tested the effectivity of a W. somnifera extract, L-asparaginase, to acute lymphoblast leukemia. L-asparaginase was first isolated by homogenizing Ashwagandha fruits. A crude protein extraction was conducted by 0.15 M KCl centrifugation at 4?C to prevent protein denaturation, with the supernatant collected. This was done three times to ensure that not most protein molecules are extracted. To purify the extracted L-asparaginase, ion exchange column chromatography was used. Briefly, the crude extract was concentrated into pellets using 40% to 60% ammonium sulfate saturation and centrifugation (9000 x g at 4?C). The pellet was dissolved in water, and subsequently loaded onto Sephadex G-25 (Pharmacia) column with 0.1 M sodium borate buffer (pH 8.6) as eluant. The eluent underwent another purification, this time using Sephadex G-75. (Pharmacia) and with the same eluant. The fractions with detected peaks were passed through another column containing CM-Sephadex C-50 with 0.01 M sodium borate buffer and ion exchange chromatographic resin. The enzyme was eluted out using a 0.1-0.5 N NaCl. Several procedures were conducted to characterize the purified enzyme. Both the subunit molecular mass (36 ? 0.5 kDa) and native protein mass (72 ? 0.5 kDa) of the purified W. somnifera L-asparaginase were determined using SDS-PAGE and Sephacryl S-400 HR (Pharmacia), respectively. The eluting buffer used in the latter was 1.0 mM phosphate buffer (pH 7.0), with a flow rate of 1.5 ml/hr. Finally, the pI of the protein was determined to be 5.0-5.5 points using 2D Gel Electrophoresis (BioRad Laboratories). Leukemia cells obtained from newly diagnosed patients were used as a source of lymphoblasts, which in turn was cultured using RPMI-1640 with 20% fetal calf serum. Cultures were maintained at 5% CO2. For the analysis of the extract’s growth inhibitory and cytotoxic activity, each of the three sets of leukocyte culture was exposed to (1) 0.1 ml phosphate buffer (negative control), (2) 2.0 IU W. somnifera L-asparaginase in 0.1 ml phosphate buffer, or (3) 1% polyhydroxyalkanoate (PHA) and 2.0 IU W. somnifera L-asparaginase. After 48 h and 72 h of incubation, viable cells were counted using a hemocytometer. To determine its growth inhibition activity, a standard MTT assay was performed. Doses of L-asparaginase used were 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, and 5 IU. Each dose of the test substance was incubated with 0.5 x 104 to 1.0 x 104 exponentially-growing lymphoblasts, after which the absorbance of the resulting mixture was measured at 550 nm. Results of MTT were expressed as leukemia cell survival (LCS), or the percent of absorbance of treated wells over untreated wells. Drug sensitivity was also computed using LC50. Statistical analyses were done using SPSS 10.0, with p < 0.05. Demonstration of the anti-oxidative mechanism of W. somnifera The relation of inflammation and resulting oxidative stress to cancer has long been recognized. Oxidative stress occurs when the amount of reactive oxygen species exceed the antioxidant materials of the body. Inflammatory processes may result to added oxidative stress that subsequently cause DNA mutations. Such mutations cause cells to replicate perpetually and unreceptive to apoptotic signals (Federico et al., 2007). This explains the efforts on identifying compounds that have anti-inflammatory and antioxidant activity for treatment against cancer. In the study conducted by Pawar et al. (2011), various measurements of antioxidant and anti-inflammation activities of Ashwagandha root extracts were performed in vitro and in vivo. The test substance was prepared by dissolving powdered root extract in distilled water, and the concentration of withanolide D per volume of aqueous extract was determined by HPTLC method to be 0.0336 % w/w. The assays used in this study were anti-lipid peroxidation activity, nitric oxide scavenging activity, hydrogen peroxide scavenging activity, and evaluation of reducing power. Except for the last assay, the values were expressed as percent of oxidation activity inhibited. Values were compared using analysis of variance (ANOVA) (p< 0.05). The anti-lipid peroxidation activity was assessed by measuring the extracts’ inhibitory effects on ferric chloride-induced polyunsaturated fatty acid (PUFA) oxidation in 10% w/v RAT colon tissue homogenate. To measure the amount of reaction that occurred, the malonyldialdehyde (MDA) resulting from PUFA oxidation was made to react with thiobarbituric acid to produce a pink red complex, which was measured at 532 nm. In measuring the anti-lipid peroxidation activity, 0.5 ml of 100-500 ?g/ml root extract was added to the cell homogenate prior to the induction of PUFA oxidation. The aqueous extract of W. somnifera roots was also tested for its nitric oxide scavenging activity that was based on the Griess-Illosvoy reaction principle. Briefly, reaction mixtures were prepared by combining 2 ml, 10 mM sodium nitroprusside, 0.5 ml phosphate buffer saline (PBS) and 2.5 ml test substance, which may be different concentrations of root extract, or methanol (negative control). A 0.5 ml aliquot of reaction mixture was then added with 1 ml Greiss reagent A, followed by the same amount of Greiss reagent B. The resulting colorimetric change was then measured at 540 nm. On the other hand, in testing for the hydrogen peroxide scavenging activity of the root extract, 0.3 ml of different concentrations of extract was added with 0.6 ml of H2O2 in PBS (pH 7.4). The absorbance of the resulting mixture was then measured at 260 nm. Finally, the ability of 250-2500 ?g/ml extract to convert Fe3+ to Fe2+ was measured. 1 ml of the extract was added with 2.5 ml phosphate buffer (pH 6.8) and 2.5 ml potassium ferricyanide. The absorbance of resulting reaction was then measured at 700 nm. Increased absorbance is indicative of increased reducing power of the extract. In the in vivo experiment, four treatment set-ups were established, (1) healthy control, (2) negative control, (3) mesalamine (positive control), and (4) test substance. Treatments were prepared as themoreversable gel that can administer 1 gm root extract/kg body weight of IBD rats per day. To prepare 20% (w/w) gel, pluronic F127 was used. For each set-up, six 200-250 g Wistar rats were used. They were maintained in ambient room temperature (25?C), and were provided with water and food ad libitum. First, a 4 mm polyethylene cannula was inserted into each of the anesthetized rat’s colon, such that its tip is only 6-8 mm away from the anus. Before the treatment, each rat underwent lavage procedure. Inflammation was induced by application of 0.25 ml TNBS in 50% (v/v) ethanol (100 mg/kg of rat weight) through the cannula. For 10 days (4th to 14th), themoreversable gels of each test substances were administered. At the end of 14th day, the rats were sacrificed through cervical dislocation. Excised colon samples were assessed for the presence and extent of inflammation and ulcer macro- and microscopically. This was noted through a scoring system, such that No ulcer, no inflammation: 0; No ulcer, local hyperemia: 1; Ulceration without hyperemia: 2; Ulceration and inflammation at one site only: 3; Ulceration and inflammation at two or more sites: 4; and Ulceration extending more than 2 cm: 5. In addition, the weight of a 10 cm segment of each of the colon samples was measured, as colitis involves a characteristic increase in colon weight due to inflammation. Histopathologically, the samples were stained with haematoxylin and eosin. In addition, MDA levels were also measured in each tissue sample. Values were expressed as mean ? standard deviation, and macroscopic and microscopic data further underwent Turkey test aside from ANOVA. RESULTS AND DATA ANALYSIS Table 1. Summary of the anti-carcinogenic activity of W. somnifera detected using various studies published from 2003 to 2011. year 2003 2010 2010 2011 Author/s Jayaprakasam et al. Choudhary et al. Oza et al. Pawar et al. Part/s of W. somnifera used leaves Aerial parts (leaves and stems) fruit root Method of extraction Silica column chromatography, MPLC, HPLC, and PTLC Methanol extraction, Silica gel chromatography Standard protein extraction, ion exchange column chromatography N/A (aqueous powdered root extract) Detected/Extracted compounds 12 withanolides and diacetylwithaferin A withaferin A, steroidal lactone and epoxide withaferin A derivatives L-asparaginase Withanolide D (detected by HPTLC) Specific activity being detected cytotoxicity Growth inhibitory and cytotoxicity Growth inhibitory and cytotoxicity antioxidative and anti-inflammatory Cancer cell line used NCI-H460 (lung), HCT-116 (colon), SF-268 (central nervous system), and MCF-7 (breast) human cancer cell lines Human lung cancer cell line NCI-H460 Human Leukemia cells and lymphoblasts Wistar rat colon cells Mode/s of testing activity MTT assay GI50 and LC50 MTT assay and LC50 antioxidative activity assays (5), in vivo colon anti-inflammatory assay Presence of activity + + + + Table 1 summarizes the relevant data gathered from the four articles. All of these studies reported to observe anticarcinogenic activity from Withania somnifera extracts. As can be seen, all the parts of the plant is a source of potent compounds that exhibit growth inhibition and cytotoxicity against various cancer cell lines. Although the study of Pawar et al. (2011) is not directed to cancer but to inflammation, as would be discussed later, its relevance to the disease is relevant in that it presents possible mechanisms by which the plant’s anticarcinogenic activity is achieved. Activity of crude methanolic extract against NCI-H460 In the study of Choudhary et al. (2010), all of the four W. somnifera-derived compounds each shown cytotoxic and growth inhibitory activities, with the lactone being the least potent, needing 6.2 ? 0.30 and 95.6 ? 2.60 ?g/ml for GI50 and LC50 respectively. On the other hand, withaferin A is the most potent of the five, needing only 0.18 ? 0.00 and 0.45 ? 0.00 ?g/ml for GI50 and LC50 respectively. No significant difference was found between its activity to that of the positive control, doxorubicin, which recorded 0.07 ? 0.00 and 0.86 ? 0.01 ?g/ml for GI50 and LC50 respectively. Anticarcinogenic activity of W. somnifera withanolides Most withanolides extracted by Jayaprakasam et al. (2003) had a dose-dependent activity. Generally, as verified in the study by Choudhary et al. (2010), among the different cell lines, lung cancer cell line NCI-H460 is the most inhibited. In addition, colon cancer cells HCT-116 were the least affected by the plant compounds. Withaferin A were the most potent withanolide against lung, colon, CNS, and breast cancer cell lines. IC50 were detected only at 7.38 to 11.6 ?g/ml against the cancer cell lines. Its derivative, diacetylwithaferin A, exhibited a similar activity, as elucidated in the MTT assay. However, not all withaferin A derivatives are as potent as its source withanolide, since 2,3-dihydrowithaferin A were observed to have decreased activity. Moreover, the physagulin D withanolides did not cause significant growth inhibitory and cytotoxic activity even at the highest dose used in the cancer cell lines used. In general, ten of these twelve were able to induce growth inhibitory and cytotoxicity effects, and when their structures were elucidated, certain features that contribute or lessen the activity of the compounds were observed. First, the presence of a double bond in withaferin A greatly contributes to its potency. Secondly, the sugar moiety at C-27 decreases the activity of the compound. Third, the hydroxyl group at C-4 increases the antiproliferative activity. Fourth, the lack of hydroxyl at C-27 was observed among tested withanolides with weak potency. Finally, a lactone ring in the structure decreases the activity. From these descriptions it can be concluded that potent withanolides are those that have the structural capacity to interact with other compounds, such that they have polar parts of their structure. In contrast, those withanolides that have non-polar moieties exposed to the surrounding biomolecules are generally weak in their anti-cancer activity. W. somnifera L-asparaginase The results of SDS-PAGE and column chromatography suggest confirm the identity of the isolated extract, L-asparaginase. The homodimeric structure of the compound was clearly elucidated by the subunit and native protein masses obtained in the research by Oza et al. (2010). In the detection of the growth inhibitory activity of W. somnifera L-asparaginase, a significant decrease in the rate of division of leukocytes was observed. After 48 hrs, cell culture from leukemia patients have the highest cell density in all the treatment groups (48 x 105 cells/ml). After 72 hrs, the cell count of untreated leukemia cells (5 x 106 cells/ml) were more than ten times the 48-hr cell count. The rate of cancer cell replication was observed to be significantly greater than the rate of normal cell replication, which causes an almost 100% increase in cell density from 48- (24 x 103 cells/ml) to 72-hr cell count (47 x 103 cells/ml). L-asparaginase, when added to the culture media, caused a significant decrease in the cell count for both normal (48 hr: 10 x 102; 72 hr: 12 x 102 cells/ml) and leukemia cells (48 hr: 75 x 103; 72 hr: 88 x 103 cells/ml), thus inferring a non-discriminating cytotoxicity. In addition, it causes a non-discriminating growth inhibition, as the rate of growth from 48- to 72-hr for both normal and cancer cells treated with L-asparaginase decreased significantly. In the results of the MTT assay, the cytotoxic activity of W. somnifera L-asparaginase against leukemia cells was found to be dose-dependent. After 24 hr of test substance exposure, the cell viability were 100±07 (0), 98±03 (0.01), 98±3.2 (0.05), 96±03 (0.1), 94±1.5 (0.2), 88±2.8 (0.5), 74±3.2 (1), 25±2.6 (2), 16±2.5 (3), and 08±0.2 (5 IU). Upon calculation, LC50 was determined to be 1.45±0.05 IU. Demonstration of its anti-oxidative mechanism In the assays performed by Pawar et al. (2011), the antioxidant activity of W. somnifera were clearly demonstrated. Upon measurement of absorption, it was found that although the root extract reduces lipid peroxidation, and its effects increases with increasing concentration, it is not as potent as ascorbic acid, a proven natural antioxidant (p=0.04). On the other hand, the scavenging activity of W. sonmifera was found to be optimum at 200 ?g/ml, which is comparable to that of the positive control, curcumin (from curry (Curcuma longa) plant), a proven natural antioxidant. Similar to the results of anti-lipid peroxidation and NO scavenging, peroxide radical scavenging activity of W. somnifera is concentration-dependent. However, the activity of 500 ?g/ml root extract (81.79%) is significantly lower than 10 ?g/ml ascorbic acid. Finally, the reducing power of W. somnifera was found to be dose-dependent. But, unlike its peroxide radical scavenging activity, its reducing power is now comparable to that of ascorbic acid. In the in vivo testing of the experiment, the root extracts were able to normalize the mucosa of the colon, as the test substance treatment group had normal crypts and lamina propria. In addition, it performed better than mesalamine, because the colon samples of the rats in positive control treatment group had distortions, edema, and traces of inflammation. However, no significant changes in the weight of the colon between treatment groups were observed. Meanwhile, the results in the measurement of MDA levels is that as it is in vitro, W. somnifera was able to decrease lipid per oxidation in vivo. DISCUSSION As mentioned earlier, inflammation and oxidative stress are major contributing factors leading to cancer. Because several studies have already confirmed the anti-cancer activity of W. somnifera extracts, it was only appropriate to determine the mechanisms behind its activity. The findings of Pawar et al. (2011), that W. somnifera decreases inflammation due to its antioxidant property, which is almost comparable to substances that are already proven anti-inflammatory and anti-oxidant compounds, is significant in establishing the mechanisms by which the plant becomes anti-carcinogenic. These antioxidants inhibit the reactive oxygen species-mediated activation of NF-kB, which regulates the genes responsible for immune and inflammatory activity (Choudhary et al., 2010; Pawar et al., 2011). The added reactive oxygen species resulting from NF-kB-induced inflammatory processes reacts with various biomolecules, including DNA. Damages in DNA that can result from these oxidants include single- or double-stranded breaks, purine-pyrimidine shifts, and cross linking. These, in turn, cause arrest or induction of replication or transcription and signal transduction pathways, all leading to perpetual division of cells observed in cancer (Federico et al., 2007). Because many of the active compounds isolated from Withania somnifera are withanolides, one of the probable mechanisms by which withanolides inhibit perpetual cell division is through scavenging reactive oxygen species that lead to cancer-causing DNA damages. In such cases, it may be valid to verify such claims. Once verified, it might be more valid if the positive control to be used in establishing the potency of withanolides is a proven substance that acts in the same manner as withanolides, that they also prevent cancer by decreasing the amounts of reactive oxygen species present in the body. One reason behind the interest on the discovery of active W. somnifera products is the toxicity of the currently used pharmacological substances. For example, prokaryote-derived L-asparaginase has recorded side effects such as anaphylaxis, pancreatitis, diabetes, seizures and blood coagulation abnormalities (Oza et al., 2010). However, as observed in the study, L-asparaginase from W. somnifera can potentially cause side effects, as it can also cause cytotoxicity to normal cells as well. The mechanism by which the L-asparaginase causes its side effects stems from its main function of converting the amino acid L-asparagine to aspartic acid. As a result, it depletes the amino acid present needed for protein synthesis. Moreover, the coagulating factors become absent, causing the various adverse effects observed from individuals treated with L-asparaginase. However, it is the leukemia cells which are more affected by the presence of L-asparaginase as they rely on outside sources for the L-asparagine they use for protein synthesis (Corm et al., 2007). Withanolides are one of the major compounds extracted from Withania somnifera. Because of their number and the observed efficacy in herbal medicine, W. somnifera has been tapped by several research groups as a source of active compounds. Such study that looked into the anticarcinogenic activity of various compounds from the plant is Jayaprakasam et al. (2003), which was able to isolate 12 withanolides from the plant. The methods used in the studies featured in this paper are commonly used in cancer research. However, the in vitro nature of these methods downplay the several factors present once the activity is assessed in vivo. Since the activity of these compounds was already shown against various cancer cell lines in vitro, withanolides are strong candidates for in vivo testing or novel chemotherapeutic treatment for patients. As far as this researcher was able to look into, there are still no studies exploring such potentials is present. The future of withanolides as a therapeutic drug against cancer is bright as, compared to the current therapeutic agents, its side effects are less because it only acts as an antioxidant. However, its potency compared to the highly potent, catatonic therapeutic agents used clinically is yet to be tested. Another suggestion for future studies is further isolation of other compounds in W. somnifera. As it was already shown by Oza et al. (2010), not only the vegetative parts of the plant are good sources of active compounds, but a component of the reproductive part as well. Probably containing previously-untapped compounds are its flowers. One compound that is interesting to discover from this plant is the one which acts on the genetic material, such as doxorubicin. In such cases, this researcher believes that the toxicity of such a compound will be less than that which prevents protein synthesis in general, which is how L-asparaginase works. In addition, the production of synthetic compounds may derive from W. somnifera compounds such as withanolides and L-asparaginase. Since Jayaprakasam et al. (2003) already shown parts of the structure that determine whether a member of withanolide family is potent, future studies can aim to make derivatives of withanolides, making them more potent based on those structure descriptions given. Because of its non-discriminating toxicity, synthetic biochemists may look into producing a synthetic compound that has a structure similar to asparaginase but is less toxic and/or more potent. References Choudhary, M.I., Hussain, S., Yousuf, S., Ahsana Dar, Mudassar, and Atta-ur-Rahman, 2010. Chlorinated and diepoxy withanolides from Withania somnifera and their cytotoxic effects against human lung cancer cell line. Phytochemistry, 71, pp. 2205-2209. Corm, S., Renneville, A., Rad-Quesnel, E., Grardel, N., Preudhomme, C., and Quesnel, B., 2007. In?uence of two different regimens of concomitant treatment with asparaginase and dexamethasone on hemostasis in childhood acute lymphoblastic leukemia. Leukemia, 21, pp. 2377-2380. Federico, A., Morgillo, F., Tuccillo, C., Ciardiello F., and Loguercio C., 2007. Chronic inflammation and oxidative stress in human carcinogenesis. International Journal of Cancer, 121, pp. 2381-2386. Jayaprakasam, B., Zhang, Y., Seeram N.P., and Nair, M.G., 2003. Growth inhibition of human tumor cell lines by withanolides from Withania somnifera leaves. Life Sciences, 74, pp. 125-132. Oza V.P., Parmar, P.P., Kumar, S., and Subramanian, R.B., 2010. Anticancer Properties of Highly Purified L-Asparaginase from Withania somnifera L. against Acute Lymphoblastic Leukemia. Applied Biochemistry and Biotechnology,160, pp. 1833-1840. Pawar, P., Gilda, S., Sharma, S., Jagtap, S., Paradkar, A., Mahadik, K., Ranjekar, P., and Harsulkar, A., 2011. Rectal gel application of Withania somnifera root extract expounds anti-inflammatory and muco-restorative activity in TNBS-induced Inflammatory Bowel Disease. BMC Complementary and Alternative Medicine, 11(34). Read More