Evaluation of anticancer activity of Plectranthus Amboinicus with Cow ark against CCl4 Induced Hepatocellular carcinoma - In Vivo.
Vinotha M and Nithya V
Pharmacognosy lab, Department of Animal Health and Management, Alagappa university, Karaikudi 630003.
Corresponding Author
Nithya V, Assistant Professor, Pharmacognosy lab, Department of Animal Health and Management, Alagappa university, Karaikudi 630003. Email: [email protected]
ABSTRACT
Back ground: Natural origin alternatives to the synthetic drugs have been found to be beneficial in curing human lungs and liver ailments. The aim of the present study designed to evaluate in vivo Anticancer effects of Cow urine distillate with Plectranthus Amboinicus (CPA), on the hematological implications, using CCL4 induced Wister albino rats model.
Methods: The indigenous cow, Bos indicus urine was distilled and characterized to know the latent compounds present in CUD, enhanced with Plectranthus Amboinicus, and its functional groups by employing HPLC and FTIR techniques. Further, In vivo anti-cancer activity was determined against HCC by evaluating HCC induced rats life span, hematological profile, such as RBC, WBC, Hb content, neutrophils, eosinophils and pellets.
Results: Cow urine with Plectranthus Amboinicus treatment significantly reduced viable cancer cell count, increased the life span of HCC inducing rats. Hematological profile retrieved to normal position in Cow urine distillate treated groups when compared with normal rat groups. All keeping in mind study suggested that cow urine may have potent anti-cancer capabilities is warranted.
Conclusions: In vivo examination, on the potential of anti cancer, paves a way for continuing, the further research ,on its unique features latent in the Scientific arena.
Keywords :Anti cancer, Life span, Red Blood Cell, Hematological profile, Cell count.
INTRODUCTION
Since the onset of the third millennium, Carcinoma has been a top most second cause of mortality in the Globe [1]. Cancer is a disease, with a common characteristic of abnormal cell division, resulting in unregulated cell growth, foray of surrounding tissues, enter into the circulation, get migrated and emerging of tumor at distant location and disrupt vital functions of the normal organs. Hepatic Carcinoma (HCC) is one of the dreadful, Gastroenterological disorders with least effective remedies. Incidence of HCC was diagnosed as 6,00,000 new cases reported in a year [2]. The treatments for liver carcinoma mainly convey on surgical liver transplantation, chemo remobilization and molecular level advanced therapy. However, such strategies are not advisable as the death rate has been still high and uncontrollable [3].
Bos indicus is reportedly known to be "Mobile Hospital" for varied incurable diseases and well evidenced through many research in India. In Veda scripts, cow urine has been used in traditional ailments for anti-taxicants and a controlling agent for respiratory dysfunction [4]. To treat cancer the cow urine, and essence of recently developed therapeutic popular drugs, were successfully reduced in the cell viability of cancer cells was reported. Moreover, Cow urine distillate (CUD) has been described as bio availability implementer for anti- cancer effects, individually or combined with anti-cancer molecules [5]. The beneficial properties of cow ark, specifically anti-cancer potential has been observed in scientific research. Among different types of Cow urine examined, indigenous Cow urine showed extreme effects [6-7]. Recent report suggested that Cow urine enhanced the ability of paclitaxel, a Taxol group against breast cancer cell lines in vitro. Many studies also convinced that Cow Urine Distillate with plant extracts improves the efficiency of phagocyte activity of microphages, present in the cell to defense against the infectious pathogens
A mountain of evidence envisaged that natural origin from plants and animal sources are still widely used for well being of human health and 50% of total therapeutic drugs
Annals of R.S.C.B., ISSN: 1583-6258, Vol. 25, Issue 4, 2021, Pages. 19406- 19414 Received 05 March 2021; Accepted 01 April 2021.
to prevent cancer associated with synthetic compound effects, in specific, used in the treatment of lungs and liver [9]. Therefore in the present investigation we report the effects of indigenous cow urine distillate with Plectranthus Amboinicus on the anti cancer potential in vivo, using CCL4 induced Wister rats animal model with hematological examinations..
Materials and Methods
Cow Urine distillate characterization
Collection of indigenous cow urine (Bos indicus)
The healthy cows have been lodged in a mutt, belonging to the Ramakrishna Mission, Mandaveli, Chennai, Tamil Nadu State. In the early morning, prior to sunrise, a second flow of urine was captured in an airtight lid container and fetched to the laboratory and filtered with filter-paper, allowing the distillation process with a simple laboratorial set up and extracted with Plectranthus Amboinicus plant extract freshly.
HPLC Analysis
10L of CPA was processed and distilled upto 17 hours continuously at 40-50 Deg. C.
to get 8L of distillate. The profile of cow urine distillate was detected, by employing C-18 column HPLC instrument with Agilent, and water was used as a mobile phase. Acetonitrile was adapted with a 1.0 ml flow per minute. Identification mark was adjusted at 230 nm Det.A ChJ /230 nm. As a result, among, obtained fractionates aqueous fraction was shown to be effective that was observed in other activity (data not shown) involving ethyl acetate. This fraction was chosen for the assessment of effects of HCC in vivo assays.
FTIR Analysis
The spectra are observed using Fourier Transform Infra-Red Spectroscopy of selected effective fraction (Elmer, FTIR Spectrum 400), annexed with a data processor. A tiny sample was converted into a small size of balls, by mixing KBr and it was compressed to form a thin film. The recorded transmittance spectra upon wave number, which exhibited with a range of 4000-400 cm-1. Based on the protocol, described by El-Naggar et al, [10]
chemical bond nature and functional groups of CUD were identified.
Determination of HCC in in vivo strategy Sample selection
Healthy Wister rats, inbred, with an average weight of 125-135 gram (N=40) were involved in the present investigation. Institutional Animal Ethical Committee Guidelines at PRIST University, Thanjavur, Tamil Nadu (743/03/abc/CPCSEA dt. 3.3.03) Required feed with proportionate nutritious value with ad libitum of water (Fig-1).
Table -1 The animals were classified into four groups such as
Group-I Treated as standard (Control)
Group-II HCC induced group, stimulated by Carbon tetrachloride Group-
III
HCC induced group I which received cow ark with Plectranthus Amboinicus a concentration of 100ml /kg. body weight.
Group- IV
HCC induced Group II animals, administered with cow ark with Plectranthus Amboinicus a concentration of 200ml /kg body weight.
Hematological Intervention Validity of cancer cells counting
Experimental rats were sacrificed and ascitic fluid was collected from the peritoneal cavity of the animal using white blood cell (WBC) Pipette and distilled thoroughly in PBS, a buffer solution [12]. A few drops of diluted suspension were kept on the Neubauer counting chamber and cells were added with tryphan blue (0.4% in normal saline). The cells could not be stained, indicated viable and those cells absorbed dye were known to be non-viable. The counts were made using the following formula.
Cell count = Number of cells X dilution factor / Area X Thickness of liquid film Determination of Life Span (ILS) / Percentage of Survival Time (SLT)
Non-viability was observed by noting the percentage increase or extend in life span and medium survival time was determined by the following equation :
Annals of R.S.C.B., ISSN: 1583-6258, Vol. 25, Issue 4, 2021, Pages. 19406- 19414 Received 05 March 2021; Accepted 01 April 2021.
Wherein survival time = day of first mortality + day of last mortality / 2 (Time is indicated by days)
Assessment of Hematological Determinants
The determination of Hemoglobin (Hb) Red Blood cell (RBC) and WBC numbers and Neutrophils content, eosinophils and pellets were counted. A varied method in counting was employed by using Leishman Stained Blood Smears [12].
Statistical Analysis
The findings denoted with values, which are expressed as Mean values. The analysis was carried out using SPSS Statistical Software. P < 0.05 was considered to be significant values.
RESULTS
High Power liquid chromatography (HPLC)Analysis.
The chromatogram showed the profiles of indigenous cow ark with Plectranthus Amboinicus, with conspicuous three sharp peaks appeared. The retention time of eluting these peaks were noted as 1.557, 1.634 and 2.113nm, indicating the nature of detected compounds. Evidence in many studies, revealed that alteration or fluctuation in the retention time (RT) is directly related with inverse square of temperature. Results elucidated that occurrence of the hexane, which eluted at 2.113 nm and other eluted peaks with their RT were noticed in the Table. A standard solution was employed for the comparison (Ethyl Acetate) and it was observed and eluted at 1.317nm.
Fig.2 HPLC profile of Cow Urine Distillate with Plectranthus Amboinicus showing 3 prominent peak indicating latent compound
The standard eluting factors are also involved in the examined distillate trials. The height of the peak indicates the latent compound, an important to observe that occurrence of hexanes in abundance may influence the therapeutic potential of Cow Distillate with Plectranthus Amboinicus.
FTIR study
The effective bio-active fraction of cow urine was investigated to know the functional groups of compounds, using absorption of infra-red spectrum. The functional groups were detected, based on their peaks eluted, which were noted in between the range of 400-4000 cm-1. Results showed that appearance of inter molecular bended amide, phosphate, alcohol, iron, a powerful and median bending of Alkenes, Alkile, halide and polysulfide stretches were recorded (Fig-3)
Annals of R.S.C.B., ISSN: 1583-6258, Vol. 25, Issue 4, 2021, Pages. 19406- 19414 Received 05 March 2021; Accepted 01 April 2021.
During the tenure of the investigation, animals were monitored separately, once a half an hour, special attention was due given while the initial 4 hours, and regularly observed for two weeks’ time. Examined rats were carefully observed once twice a day in order to note the symptoms of toxicity, survival behavior of the animals [11]. The animals were permitted to acclimatize prior to the laboratory condition for a week.
Viable Cell count for Life Span Time
Count of viable cancer cells reported that however, it enhanced the non-viable cancer cells count, median survival time (MST) has increased to 24.72. (Percentage of increased life span time = 19.35%) and 33.46 (Percentage of ILST = 49.28 ) while, the administration of cow urine distillates to the HCC 1 and HCC II Group animals respectively. At the dose of 100ml. body wt. and 200ml /kg body wt. and the reference of bleomycin, indicated survival time 20.12 and 38.17 (percentage of ILS = 88 respectively)
Hematological determinants
The determinants of the hematological studies on the HCC I & II induced rats were observed to be significant variation at (P < 0.05), when compared to the control. Moreover, a remarkable reduction in Hemoglobin content (Hb) Red Blood cells, in relating to the group receiving CCL4 induced HCC. Furthermore, Neutrophils’ (Table -2 and Fig. 4), eosinophils, and pellets were detected in decline in the CCL4 induced rats when compared with normal saline group (Control). Evidently, cow ark (100ml/kg body Wt.), and (200ml/kg b.wt) have significantly fluctuated those alteration and revert to normal level.
Table 2-Effects of Cow Ark with Plectranthus Amboinicus on the hematological indicators in HCC induced Wistar Rats
Group Hb RB
C
WBC COUNT
Neutrop hil
Eosinophi l
Saline (Control) 12.84 7.48 4.35 0.19 0.16
CCL4 induced group 3.21* 1.96 2.67 0.9 0.11
HCC 1 Group 100ml/kg b Wt.
9.17 2.87 3.51 0.5 0.12
HCC 2 Group 200ml/kg b Wt.
11.36*
*
6.92 5.02 0.17 0.14.8
The Table indicates the Mean value of triplicate n=6. p < 0.05 compared with control group
Hb =Hemoglobin RBC = Red Blood cells WBC = White Blood cells
Fig 4 Hematological studies on the HCC I & II induced rats DISCUSSIONS
Using HPLC analysis, cow urine distillate with Plectranthus Amboinicus was segregated into three fractions. Among them effective fraction was identified based on their effectiveness of inhibitory potential of biological activity and was believed to be a responsible factor for the varied biological properties of cow urine distillate. The attenuating characteristic of cow urine is found to be occurrence of few volatile and non-volatile constituents [13]. FTIR analysis elucidated that in examined cow urine, presence of amide, alkene, alcohol, and polysulfide groups recorded, were found in good agreement with early findings of Widyamto et al[14] who detected, the occurrence of amide and CH3 broad bending in cow urine.
A present in vivo study revealed that, increase life span of cow urine distillate treated animal groups at 200ml/kg obtained might be due to the decline of nutritional fluid volume, in the cancer cells, resulting in late cell division and the similar findings were observed in earlier study [15]. A drastic decline in the viable cell count and an enhancement in non- viable cell count directly correlated with protective role of anti-cancer potential of cow urine distillate with Plectranthus Amboinicus in Rats.
Anemia has been frequently observed in HCC cancer patients due to iron deficiency, by the process of hemolytic condition, resulting in a decline of RBC counts (Gupta et al.,2004). Treating with cow urine distillate (100ml/kg) b.wt. retrieved the RBC counts progressively. Similarly WBC count fluctuate and retrieved to the normal condition while treated with Cow urine distillate, indicating hematic protective impact, by unaltering myelotoxicity, an often occurred side effects of the chemotherapy [13].
Animal estrus urine having rich sources of pheromones and Pheromone compound for the potentials of many diseases. [16-19]. Furthermore, there have been many effective drugs for antimalarial, such as, quinine, CLQ, and other classes of drugs compounds are in use, specifically plant based, and animal origin template compounds for the formulation are affordable [20-21]. Enormous accounts of studies have been showed that free radicals implicates against disorders covering diabetes, cancer, antidiarrheal, antiiflammatory, antimicrobial, antibiofilm and wound healing [20-24]. In the present study heavy doses of CUD decline cancer cell viability, fluctuating and restore the hematological profile and increased life span, compared with control rats were detected.
CONCLUSION
Innovative findings indicated that the cow urine distillate Plectranthus Amboinicus has promising anti-cancer activity, as demonstrated by in vivo experiment. Further research is too suggested to isolate the responsible compounds for the anticancer activity to evolve a
Annals of R.S.C.B., ISSN: 1583-6258, Vol. 25, Issue 4, 2021, Pages. 19406- 19414 Received 05 March 2021; Accepted 01 April 2021.
Authors thankful to authorities of the Alagappa University, Karaikudi, Tamil Nadu, India, which was supported the research work done by the MHRD-RUSA 2.0-F.24/51/2014-U, policy (TNMulti-Gen), Dept. of Edn. Govt. of India.
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