Evaluating the Immunomodulatory and Detoxifying role of Tinospora cordifolia and Solanum nigrum in the treatment of Colorectal Cancer
Alkeshkumar Patel1, Hemangini Vora2, Neeraj Jain3, Manish Nandpal1,
Sahil Panjvani4, Samir Patel1*
1Ramanbhai Patel College of Pharmacy, CHARUSAT, Changa, Gujarat, India.
2Gujarat Cancer and Research Institute, Ahmedabad, Gujarat, India.
3PD Patel Institute of Applied Science, CHARUSAT, Changa, Gujarat, India.
4Vadodara Clinical Laboratory, Vadodara, Gujarat.
*Corresponding Author E-mail: samirpatel.ph@charusat.ac.in
ABSTRACT:
Background: Colorectal Cancer may occur due to environmental pollutants like polycyclic aromatic hydrocarbons and hydrazines. These are indirect carcinogens and initiate carcinogenesis after their metabolic transformation within the body. This causes more than 0.94 million deaths every year. Methods: We investigated the prophylactic intervention of hydroalcoholic extract of Tinospora cordifoilia stems and aqueous extract Solanum nigrum leaves in 1, 2 dimethyl hydrazine induced colorectal cancer in Sprague Dawley rats. The chemopreventive potential was observed at low and high doses for 27- and 32-weeks duration. The chemopreventive potential of natural products was evaluated by estimating the change in body weight, diarrhoea, hematological parameters, change in cytokines level, cancer stem cells marker ALDH1,T lymphocyte subpopulation estimation by flow cytometry, confirmation of adenoma, adenocarcinoma and liver metastasis based on histology. Results: The high dose of the test drug showed a significant difference (p<0.05) compared to disease control groups animals with respect to blood and tissue-based parameters. The high-performance liquid chromatography analysis of herbal extract showed the presence of berberine, tinosporide, quercetin,ellagic acid and naringenin in natural products. Probable mechanisms involved downregulating mRNA of nuclear factor kappa B, matrix metalloproteinases-9, tumor necrosis factor-α, interleukin-6 and cyclooxygenase-2 while promoting proapoptotic caspases, antioxidant capacity, interleukin-12. Conclusion: The high dose of the test drug has chemopreventive and detoxifying potential against carcinogen induced colorectal cancer in animal model.
KEYWORDS: Natural products, Colorectal cancer, Metabolic transformation, Cancer stem cells, Detoxification.
INTRODUCTION:
Colorectal Cancer (CRC) is the world’s third most predominate and second lethal cancer. As per the GLOBOCAN, there are more than 20 million new cases of CRC detected at present and it may jump to more than 31 million by the end of 20401.
The multiple hallmarks of cancer compromise the body’s defense with respect to immunosurveillance, mitochondrial energy production, angiogenesis, apoptosis and telomerase functioning2. The post metastasis five years survival rate for CRC is very low compared to other cancers3. There are multiple somatic variants (KRAS, BRAF, EFGR, NRAS), mismatch repair deficiency and microsatellite instability involved in the CRC development and progression4. Recent clinical trials were conducted on Aspirin and Metformin as chemo preventive agents for CRC. The randomized trial concluded CRC as preventive disease, but it would be risky to continue these chemo preventive agents for chronic period5. The presence of aberrant crypt foci (ACF) and adenoma can be considered as initial stage biomarkers to judge the progression of the disease6. As per the Agency for toxic substances and disease registry (ATSDR, USA), polycyclic aromatic hydrocarbon (PCAH), hydrazines, and nitrosamines are environmental carcinogens. People may come under exposure to these chemicals during the occupation, tobacco product use, certain foods, or accidental leaks from the manufacturing units. The first-line agents are 5-fluorouracil (5-FU), irinotecan, panitumumab, bevacizumab and cetuximab. Recently, the multiple kinase inhibitor, Regorafenib was discovered as a small molecule to treat the advanced stages of CRC7. But the issues of resistance, recurrence and aggressiveness remain as it is. That leads to treatment failure and mortality8. The possible contributors are Cancer Stem Cells (CSC), tumor microenvironment with poorly differentiated heterogeneous population, Wingless-related integration site (Wnt) signaling, Warburg effect, DNA damage repair mechanism and ATP-binding cassette (ABC) transporters for drug elimination from tumor cells9.
The chemopreventive agents exert their role by either blocking cancer initiation (inhibiting critical interactions of carcinogens with their cellular targets) or by suppressing action (hampering cancer promotion and metastasis)10. The beneficial value that works for chemoprevention involved antioxidant, immunomodulatory, nutraceutical, indirect carcinogen metabolism suppressor and mucosal protective properties11. Curcumin, luteolin, resveratrol and genistein have been tested in clinical trials for their detoxification property. Natural products offer the advantage of a safety margin with high anticancer efficacy by targeting multiple targets. Curcumin has shown its chemoprotective potential against CRC, but it has major drawbacks with respect to Pharmacokinetics, chemical instability and bioavailability. Tinospora cordifolia and Solanum nigrum have vital phytoconstituents that work at multiple cancer hallmarks. The possible signaling pathway switched on by the above phytoconstituents is Nrf2/KEAP1 which further promotes the synthesis of cellular antioxidants like superoxide dismutase, glutathione, heme oxygenase and catalase12. The 1, 2 dimethyl hydrazine (DMH) is an indirect carcinogen. After its metabolic transformation through glucuronidation reaction in the liver to potent alkylating metabolites called methyl carbonium ion, DMH activates Clear factor kappa‑light‑chain enhancer of activated B‑cells (NF‑κB), Kirsten rat sarcoma viral oncogene homolog (KRAS) and Transforming growth factor beta (TGF-β), Wnt13–15.
Cancer stem cells (CSC) are considered recent targets for the novel CRC treatment. It plays a critical role in treatment resistance, recurrence and metastasis16. The presence of different surface markers (ALDH1, CD133, CD44, CD166) helps to predict disease-free survival and overall survival post-treatment the higher expression indicates invasiveness, metastasis and disease aggressiveness17,18. ALDH1 and CD44 have been targeted to evaluate the chem-preventive potential of dietary phytoconstituents in MCF-7 cell lines mammospheres19 by Immunohistochemistry (IHC) 20. Tumor infiltration and systemic immune subpopulation positively correlated with CRC progression21. CD4+T, CD8+T and natural killer (NK) cells are used by cancer biologists to determine the prognosis of CRC22,23. Cytokines play crucial roles in the CRC fate. The IL-12 can be used to stimulate antitumor immunity through immunomodulatory CD8+ T and NK cells in CRC. The TNF-α is believed to be a promoter for CRC invasiveness, metastasis and migration through upsurge tumor-associated calcium signal transduction protein (TROP)‑2. The activation of signal transducer and activator of transcription (STAT3) by IL-6 induces epithelial‑to‑mesenchymal transition (EMT), tumor plasticity and cancer stemness24.
MATERIALS AND METHODS:
Preparation of herbal extract, use of chemicals, kits, and instruments during the study:
The freeze-dried hydroalcoholic (30:70) extract Tinospora cordifolia (TC) was received as a gift sample from Pharmanza herbals pvt ltd, Dharmaj, Gujarat, INDIA. The shadow-dried leaves of Solanum nigrum (SN) were used to prepare aqueous extract by cold maceration technique. Preliminary phytochemical screening and HPLC were carried out to qualitatively analyze the presence of phytoconstituents. The carcinogen DMH was bought from Sigma Aldrich. The changes in the cytokines (IL-6, IL-12, TNF-α) level in plasma were determined using Enzyme‑linked immunosorbent assay (ELISA) kits manufactured by Elabscience, USA. Complete blood count (CBC) was estimated by aMindray BC-5130 analyzer. The epoch microplate spectrophotometer plate reader (bio tek instrument, USA) was used to estimate each cytokine level in plasma. The surface markers for immune subpopulation cells (CD4, CD8, NK cells) were purchased from BD Biosciences. The same cells were estimated by BD FACS Canto 10 colour flow cytometer (BD Diva software). CSC markers ALDH1 were purchased from Invitrogen. The IHC was done by using an auto immunostainer instrument, Ventana Benchmark XT (Ventana Medical Biosystems, USA) and Ventana reagents as per the manufacturer’s protocol. Images of colon histology and IHC were captured by an inverted trinocular microscope (Carl Zeiss, axiovert ALFL).
Phytochemical evaluation by High-Performance Liquid Chromatography (HPLC):
For the HPLC technique, 10mg of all the six standards (Berberine, tinosporide, kaempferol, naringenin, quercetin, and ellagic acid) were accurately weighed and transferred to a 10mL volumetric flask separately and marked up the volume using methanol (1000µg/mL). Working stock solutions were prepared by taking 2mL standard stock solution (1000µg/mL) of each drug transferred to a 10mL volumetric flask separately and marking up the volume using methanol to yield a working stock solution containing 200µg/mL. Preparation of sample solutions for TN and SN was carried out by taking accurately weighed 15mg of extract and transferred it to a 10mL volumetric flask, then the content dissolved in 5mL methanol with the aid of sonication and the final volume was makeup to mark with methanol (1500µg/mL).The mobile phase was prepared by taking 70 volumes of 0.1% Formic acid and 30 volumes of Acetonitrile, respectively (70:30% v/v) then filtering it using a 0.45µm membrane filter and sonicate to degas the mixture.
An analytical Reverse Phase Liquid Chromatography method has been developed for the simultaneous estimation of Ellagic acid, Berberine Hydrochloride, Quercetin, Tinosporide, Naringenin and Kaempferol from SN and TC extracts. Water AllianceTM HPLC system (Waters e2695 separation module) coupled with a Photodiode-Array detector (2998 PDA) and an auto-injector were used for HPLC analysis. The system was monitored by Empower III software. The chromatographic separation and detection were achieved using Luna ® C18 column (250 mm ×4.6 mm, 100 Å; 5 μm) as a stationary phase, an isocratic elution having mobile phase 0.1% Formic acid: Acetonitrile (70:30 % v/v) at a flow rate of 0.4mL/min, with an oven temperature to be set 30şC.
Experimental Pharmacology:
A total of forty-two healthy male Sprague-Dawley (SD) rats were divided into four groups. The animal study protocol (RPCP/IAEC/2019-20/R2) was approved by the institutional animal ethical committee. The CPCSEA guidelines were strictly followed to perform animal experiments. The animal study was modified and prepared in alignment with ARRIVE guidelines (The ARRIVE Essential 10 and The Recommended Set). Animals were divided into normal control (NC; n=6), disease control (DC; n=12), prophylaxis test high dose (PTH; n=12) and prophylaxis test low dose (PLH; n=12). All the blood and tissue-based parameters were estimated for each group of animals by sacrificing half the population (n=6) at the end of the 27th week.The metastasis, overall survival, metastasis-free survival and tumor multiplicity that was determined at the end of the 32nd week (remaining six rats). CRC was induced by subcutaneous injection of DMH in all rats except normal control at the dose of 20mg/kg for first 10 weeks and 30 mg/kg for another ten weeks (total carcinogenesis induction period was 20 weeks). Both test drugs at the dose of 50mg/kg (PTL) and 100mg/kg (PTH) were administered 1 week before, during and 1 week after completion of the DMH administration by oral route.
Assessment of morphological changes:
Change in body weight due to muscle wasting or cachexia was estimated at the end of the 27th week from each group compared to the initial weight. Diarrhoea can be scored as per the stool consistency (score; 0= absent, 1= mild, 2= moderate, 3= severe, 4= occult blood). It helps to categorize stages of cancer due to underline mucosal inflammation, gut microbiome dysbiosis and leaky tumor secreting blood in the stool. The colon length to weight ratio (CLWR) was measured by measuring colon length in cm and dividing it by colon weight in grams. The spleen index (SI) and liver index (LI) were determined by measuring organ weight in grams and dividing it by the whole weight of the animal.
Assessment of hematological changes and cytokines level by ELISA:
The rat’s blood was withdrawn under suitable anaesthesia by a retroorbital puncture to determine red blood cell (RBC), white blood cell (WBC), platelets, and percentage of lymphocyte counts. Important cytokines to predict the effect of test drugs on carcinogenesis were estimated at the end of the 27th week from rats’ plasma. It involves tumor-suppressing IL-12 and tumor-promoting IL-6, TNF-α. It was estimated by sandwich ELISA as per the producer's instruction in 100μL of the rat’s plasma sample. Later optical densities (OD) were measured at 450nm in 10minutes.
Changes in the immune cell subpopulation:
The immunomodulatory effects of natural products were evaluated at the end of the 27th weeks. The T lymphocyte CD4+ CD8+ and CD8CD161+ T cells were counted in whole blood by flow cytometry as per the producer’s instruction along with established lab protocol. Unique immune cell surface markers were used to estimate cell population. It includes APC mouse anti-rat CD4 clone OX-35, PE mouse anti-rat CD161a clone 10/78 BV421 mouse anti-rat CD3 clone 1F4, FITC mouse anti-rat CD8a clone OX-8 and APC-Cy 7mouse anti-rat CD45 clone OX-1 from BD Biosciences.
Histology of colon and Immunohistochemistry of CSC markers- ALDH1:
After sacrificing the animals, the entire colon was isolated carefully. It was cleaned by administering normal saline inside the cavity. Then colon was fixed in 10% of the formalin solution and sent to the histopathology lab. In lab, the colon was fixed with paraffin wax and slim segments of around 4-5 micrometers were chopped with a microtome. Hematoxylin and eosin staining were used to stain the different tissues and judge the stages of malignancy.
IHC was performed as per the established lab protocol. Initially, the fine sections of tumor tissue were sliced using a microtome. Later it was affixed on 3-Aminopropyl triethoxysilane (APES) coated slides. Then deparaffinization was carried out with EZ prep solution followed by antigen retrieval using cell conditioning solution (CC1) at 95°C. This step was followed by sequential steps of slide incubation with different reagents. First with optiview peroxidase inhibitor for 4 minutes followed by optiview HQ linker for 8 minutes and 100µl of primary antibody of ALDH1. The antigen-antibody conjugate was identified by 3,3’-diaminobenzidine (DAB) and counterstained with hematoxylin. The IHC carries the advantage of extraordinary specificity and sensitivity. Based on CSC marker (ALDH1) expression, %positivity can be reported to judge the conversion of normal stem cells to CSC 25.
Tumor multiplicity, overall survival and metastasis determination:
At the end of the 27th week of the study, out of the total 12 rats, half were sacrificed (n=6). The remaining 6 rats were kept alive without any treatment and sacrificed at the end of the 32nd week. At this point, animals were observed for tumor multiplicity, overall survival and spread metastasis to vital organs like the liver, lungs and visceral cavity.
Statistical analysis:
All the results were stated in Mean±SEM. All the parameters were statistically evaluated with one-way ANOVA followed by Tukey's multiple comparison tests, Mann- Whitney (tumor multiplicity) and Kruskal Wallis test (for scoring) by applying graph pad prism software. The P<0.05 believes as a significant difference between the groups.
RESULTS:
Phytochemical evaluation with HPLC:
The HPLC system detected the presence of berberine and tinosporidefrom the TC extract while naringenin, quercetin and ellagic acid were found in the SN extract. We couldn’t find the presence of kaempferol with this experimental setup (figure 1).
Figure 1: HPLC chromatogram of herbal extract. An analytical Reverse Phase Liquid Chromatography method has been developed for the simultaneous estimation of Eilagic acid, Berberine Hydrochloride, Quercetin, Tinosporide, Naringenin and Kaempferol. a: blank, b: standard mixture at 254 nm, c: standard mixture at 210 nm, d: berberine peak, e: phytoconstitutents from SN, f: tinosporide peak
Figure 2: Morphological changes at the end of the 27th week in between the different groups. a: Change in body weight as revealing presence of the cachexia, b: severity of diarrhoea as indicator of colitis, c: colon length to weight ratio for predicting microadenoma and adenoma, d: morphology of colone length to weight ratio, e: liver index values to understand abnormal liver changes during carcinogenesis, f: spleen index values to indieates hematological changes. All values are expressed as mean ± SEM of 6 animals. a, P< 0.05 versus disease control, One-way ANOVA followed by tukey’s multiple comparison tests using GraphPad Prism
Morphological changes:
The body weight significantly reduced (p<0.05) in the DC group compared to all other groups. The PTH has protected rats against loss of body weight. It remains the same as NC during the study (figure 2a). The highest score of diarrheas was found in DC compared to all other groups due to the presence of occult blood in the stool. The PTL was unable to protect against colitis-induced mild diarrhea while PTH prevented the occurrence of diarrhea (figure 2b). The presence of micro tumors and developing adenoma, and adenocarcinoma decrease the length of the colon but increased the weight in the DC group rats which a showed a significant difference (p<0.05) compared to all other groups. The PTL was unable to maintain the normal value of CLWR like PTH (figure 2c, 2d). The LI values of DC showed significant difference (p<0.05) compared to NC but not with PTH and PTL (figure 2e). The SI values of the DC group showed significance difference (p<0.05) compared to all other groups (figure 2f).
Hematological and Cytokines level changes
The RBC values of DC were significantly lower (p<0.05) compared to all other groups due to occult blood in the stool (figure 3a). The WBC counts were not significantly different between the groups. Anyhow high value was observed for PTH (figure 3b). The percentage of lymphocyte counts was significantly reduced (p<0.05) in DC compared to all other groups (figure 3c). The rise of lymphocyte populations in PTH and PTL due to its immunomodulatory effects worked as antitumor immunity. The PLT count was significantly high in DC rats compared to all other groups as an indicator of tumorigenesis progression (figure 3d).The level of IL-6 (figure 3f) and TNF-α(figure 3e) were found to be significantly high (p<0.05) in DC group rats compared to all other groups. Pretreatment with test drugs could prevent this cancer-promoting surge of cytokines during the study. The IL-12 (figure 3g)was found to be drastically reduced in the DC and found a significant difference (p<0.05) compared to all other groups. The PTH and PTL could overcome the tumor promoted immunosuppression and rise the value of IL-12. Anyhow these values are not comparable with NC.
Immune cells subpopulation- CD4+, CD8+ and CD8CD161+ T cells:
The percentage of immune cell populations found no significant difference (p<0.05) for CD4+T cells (figure 4a),and CD8+ Tcells (figure 4b) among all the groups. However, the CD8CD161+ T cells were found significantly reduced (p<0.05) in DC compared to all other groups (figure 4c).
Figure 4: Effects of treatment of an immune cell population estimated by flow eytometry. The test drugs improved NK cells related to the population but no significance difference was observed for CD4+T and CD8+T cells. A. Change in CD4+T cells, b. change in CD8+T cells, c. change in CD8CD161+T cells. All values are ecpressed as mean ± SEM of 6 animals. P<0.05 versus normal control, b, P < 0.05 versus disease control. One-way Anova followed by Tukey’s multiple comparison tests using GraphPad Prism.
Histology and Immunohistochemistry of CSC marker- ALDH1 (observed 10X):
The NC group rat’s colon has no spontaneous tumor development and found with normal cellular architecture. The crypts were found normal and all tissue boundaries were found normal (figure 5a). The DC group rats were found with poorly differentiated adenocarcinoma, lymph node infiltration, and breach of serosa boundary and even with the most resistant and aggressive signet ring cell carcinoma (figure 5d, 5e, 5f). The PTL group rats showed moderately differentiated adenocarcinoma (figure 5c) while PTH showed dysplasia and adenoma in few rats (figure 5b). The high positivity (figure 5i, >60%) found with DC groups rats while the NC and PTH groups animals showed low positivity (figure 5g, <10%). The PTL groups’ rat showed moderate positivity (figure 5h, 20-50%) for ALDH1.
Figure 5: Histology and immunohistochemistty results of ALDH markers of colon tissue. Test drugs preserve the normal colon tissue and restrict the carcinogenesis in the preliminary stage. Test drugs reduced the ALDH1 expression that indicates a decrease in the disease aggressiveness. a: normal colon, b: dysplasia indicates beginning of carcinogenesis, c: moderately differentiated adenocarcinoma. D: resistant signet ring cell careinoma, e: serosa infillration by tumor cells, f: lymph node metastasis, g: low positivity of ALDH1, h: moderate positivity of ALDH1, high positivity of ALDH1.
DISCUSSION:
The existing therapy for the failure of CRC due to resistance and recurrence leads to high mortality 26,27. As much as 50 per cent of CRC cases relapse and indicate a lack of sustained therapeutic potential of conventional therapy. Natural products can be used for chemoprevention and detoxification in routine life due to their nontoxic, efficacious and sustainable therapeutic values28. DMH is used for the experimental Pharmacology CRC model, but the biochemical changes are like human CRC 29. Cachexia occurs at an advanced stage of the CRC due to loss of appetite, visceral pain, enhanced metabolism, and loss of skeletal muscle. Berberine, quercetin and naringenin are believed to be responsible for protective effects against cachexia 30. Diarrhea would be due to chronic colitis31, dysregulation of aquaporin channels and gut microbiota dysbiosis32. The antioxidant33, anti-inflammatory and antiproliferative properties of the test drug could protect rats against diarrhea. Flavonoids could be the responsible phytochemicals. Colon length-to-weight ratio is directly correlated with micro and macro tumors in the colon 34. The daily dosage of natural products interferes with the action of DMH and helped the colon to detoxify it against toxic metabolites of DMH. The liver index is indicative of subtle changes in the milieu. The rise in the value in DC indicates liver metastasis35. Even the fatty changes in the liver made by DMH also raise the liver index. Natural products’phytoconstituents positively modulated systemic immunity and preserve the spleen index near normal value in treated animals36. The notorious cytokines for CRC involved IL-6 and TNF-α. The chronic surge of these cytokines further strengthens downstream pathways like wnt/β‑catenin, NF‑κB, JAK‑STAT and JAK‑MAPK. The prophylactic use of natural products controlled the release of these cytokines. The IL-12 can stimulate NK cells for its antitumor activity 37,38.
Berberine has been reported to rise the expression of IL-12 and mediate anti-inflammatory immunomodulation activities39. Total WBC counts appeared similar in all the treatment groups, but the differential counts seem different concerning the percentage lymphocyte. The population of lymphocyte works as the main soldiers to fight against malignancy, while the rise in neutrophils and monocyte may promote malignancy and nullify the test drug effects. The CD8CD161+ T cells are therapeutically highly potential cytotoxic cells with the advantage of memory has been rising after the pretreatment with natural products. High ALDH1 is an expression associated with poorly differentiated tumor and poor survival of the host. The DC group rats have shown high positivity for ALDH1 and justify the worst disease outcome compared to other groups. The SN has a well-proven record of hepatic detoxifying activity against carbon tetrachloride and other insulting agents. The probable mechanisms involved increasing glutathione S-transferase level, and downstream targets of nuclear factor erythroid 2–related factor 2 (Nrf2). The presence of polyphenols might be responsible for detoxifying the colon and liver against toxic DMH metabolites and their persistent oxidative stress. The natural products SN and TC have vital chemopreventive and detoxifying phytoconstituents like berberine, tinosporide, quercetin, ellagic acid and naringenin, β-carotene, luteolin, degalactotigonin, clerodanediterpenoids40. Clerodanediteroenes have been proven to be anti-hepatocellular carcinoma activity. So, it might have prevented liver metastasis in the test group’s rats. TC extract can modulate phase II metabolic enzymes and contribute detoxification of DMH.
CONCLUSION:
The nontoxic nature of herbal natural products can be used to prevent sporadic cases of CRC. A more robust clinical platform must be used to confirm the evidence received during this preclinical study. The susceptible people may get benefits of this natural product based on its prophylactic application which is not possible with chemotherapy and radiation.
COMPLIANCE WITH ETHICAL STANDARDS:
The animal study was conducted as per the latest Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) India guidelines.
AUTHOR CONTRIBUTION:
Alkeshkumar Patel, Samir Patel, HemanginiVoradevised the study. Alkeshkumar Patel, Darshan Patel, Neeraj Jain planned experiments. Alkeshkumar Patel, Sahil Panjwani and Manish Nandpalperform the experimental work. All authors given intellectual input and contributed to the organization of the manuscript.
FUNDING:
The funding was provided by CHARUSAT SEED Research Grant (proposal no. 17) and Pharmanza Herbals Pvt Ltd, Dharmaj, Gujarat, India.
CONFLICTS OF INTEREST:
Authors declare no conflicts of interest.
ETHICAL APPROVAL:
The animal study protocol (RPCP/IAEC/2019-20/R2) was approved by the institutional animal ethical committee (IAEC).
ACKNOWLEDGEMENTS:
This Research work was supported by Ramanbhai Patel College of Pharmacy, CHARUSAT, Changa and Gujarat Cancer and Research Institute (GCRI), Ahmedabad, INDIA.
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Received on 23.06.2023 Modified on 15.11.2023
Accepted on 08.03.2024 © RJPT All right reserved
Research J. Pharm. and Tech 2024; 17(5):2155-2163.
DOI: 10.52711/0974-360X.2024.00340