Evaluation of Antitumor Activity of Sivanar Amirtham – A Herbomineral Formulation belonging to Indian Traditional System of Medicine using Dalton's Ascites Lymphoma in Mice

 

Moushumi Baidya^1,2*, Shvetank Bhatt³, Himangshu Sekhar Maji¹, Kuntal Manna⁴, J. Anbu⁵

¹Department of Pharmaceutical Technology, JIS University, Kolkata, West Bengal, India - 700109.

²Bharat Pharmaceutical Technology, Agartala, West Tripura, 799130, India.

³School of Health Sciences and Technology,

Dr. Vishwanath Karad MIT World Peace University, Pune - 411038, India.

⁴Natural Cum Advance Synthetic Lab, Department of Pharmacy,

Tripura University (A Central University), Suryamaninagar - 799022, India.

⁵Department of Pharmacology, Faculty of Pharmacy,

M.S. Ramaiah University of Applied Sciences, MSR Nagar, Bangalore - 560054, India.

 

ABSTRACT:

The purpose of the present study is to evaluate the antitumor activity (ATA) of traditional herbal preparation Sivanar Amirtham (SA) on Dalton’s Lymphoma Ascites. Siddha medicine system (SMS) is a traditional system of medicine originated from ancient Tamilakam of South India. Siddha medicine is a traditional healing system from Tamilakam in ancient South India. For our purpose, we have performed acute toxicity (AT) study as per OECD guidelines 423 and ATA by xenograft method. In this study, a single dose of 300, 1000 and 2000 mg/kg of Sivanar Amirtham suspension (SAS) was orally (p.o.) administered in mice and animals were observed for 14 days. For antitumor study (ATS), we have used DAL cells which were intraperitoneally (i.p.) inoculateded into mouse. The ATAs were studied by monitoring the parameters such as cell growth inhibitors, tumor weight measurements, mean survival time of DAL bearing mice as well as changes in depleted haematological and biochemical parameters due to tumorigenesis. The SAS was also evaluated for in vitro cytotoxicity study in different concentration and the viability of cells was determined by exclusion method of trypan blue dye (TBD). The AT study showed no signs of toxicity and no mortality after single administration of SAS. SAS caused significant decrease in packed cell volume (PCV) (value), Tm volume (value) and viable cell count (value), and it prolonged the life span of DAL Tm carrying mice. Haematological and biochemical profiles were reverted to normal levels in SAS treated mice. The results of in vitro cytotoxicity show that SAS showed significant ATA in mice with moderate DAL levels. The IC50 value turned into discovered to be 800 μg/ml from the in vitro cytotoxicity examine. The study strongly suggests that SAS has the potential to be an antitumor medication against DAL cells induced Tm and it can be extrapolated for further cancer (CA) prevention applications.

 

 

 

INTRODUCTION: 

CA is one of the topmost causes of deaths worldwide; in many countries, it's considered as second leading reason of loss of life after heart ailment. As per World Health Organization report, by the year 2030 there will be 21 million new CA cases and 13 million CA deaths are probable to occur worldwide.

 

Normal cells turn into cancerous by continuous and uncontrolled division of cells and their growth¹. Various environmental factors, lifestyle changes or behavioural exposures are the main culprits for the initiation of CA. The main causes of CA are due to chemicals, infection, radiation, heredity, physical agents, and hormones. Chemotherapy is a potential therapeutic approach to fight against various types of CA as monotherapy or combination approach with surgery or radiation therapy but chemotherapeutic effects of most of the drugs have shown limited efficacy due to the development of multiple adverse effects and resistance². Several herbal products have been reported to exhibit significant anti-CA actions with minimal to no side effects were used in polyherbal Siddha preparations which are emerging trends in curing many CAs worldwide especially in developing countries. This work makes an attempt to screen, Siddha product against CA as they are less likely to associate with serious side effects³.

 

SA is a traditional herbo-mineral formulation used to cure various ailments in Siddha medical system (SMS). It is a combination of total nine ingredients i.e. Dryopteris filix-max, Elemental mercury (purified), Aconitum napellus, Elemental sulphur (purified), Zingiber officinale, Piper nigrum, Piper longum, Arsenic disulphide (purified), Borax (purified)⁴.

 

SMS delivers wide line of treatments for different kinds of chronic and dangerous diseases including CA. SMS is one of the traditional systems of medicine in India. Its origin can be traced back to ancient Tamilakam of South India⁵. The SMS was established about more than 2500 years before by the eminent powers known as Siddhars and hence the name Siddha Medicine⁶. Above 80% of the siddha medicines are formulated by herbal products. But in some severe diseases herbal medicines alone is not much effective. In that condition, Siddhars enumerated some herbo-metal and herbo-mineral formulations. This work attempts to evaluate some Siddha products against CA as they are less likely to produce any life-threatening adverse effects^7,8.

 

Traditionally SA is used to promote strength of bones and joints by virtue of anti-inflammatory and analgesic activity. It is also used to treat stiffness of muscles, coccyx pain, back spasm, and back injury. In addition, partially it is also useful in hypothyroidism, ankylosing spondylitis, sensory neural hearing loss and avascular necrosis⁹.

 

Recently, it has been found that an intimate relationship exists between Tm progression and inflammation. Many forms of CAs are linked with and originate from site of infection, long-term irritation, and inflammation¹⁰. Inflammatory cells which are found largely in the Tm- microenvironment play a critical role in invasion, migration, and metastasis. The link between inflammation and CA led us to find out the ATA of the anti-inflammatory Siddha drug “SA” using in vitro and in vivo preclinical study¹¹.

 

MATERIALS AND METHODS:

Animals:

Swiss albino mice (SAM), female, weighing 25-30g have been used for the AT find out about and male SAM weighing 25-30g have been used for ATS. The animals have been bred, reared, and housed in the animal residence of MSRUAS, Bangalore. The animal experimental tactics have been authorized by means of the Institutional Animal Ethics Committee of MSRUAS (IAEC certificate no: XVIII/MSRFPH/M-05/08.02.2017).

 

Acute toxicity (AT) study:

The oral AT of SAS turned into evaluated in line with OECD guidelines 423 SAM (25-30gm) have been handled with wherein the test dose limited to 2000 mg/kg, p.o. All the animals had been kept at in a single day fasting before giving dose with sufficient availability of water. The animals have been grouped into 4 groups of three animals according to organization (n = three). After an overnight fast, dealt with group were administered orally at doses of 300, 1000 and 2000 mg of SAS consistent with kg of body weight respectively^12,13. All mice had been found at 1, 2 and 4 h after treatment with test drug and periodically in the course of the first 24 h then, day by day till 14 days for mortality or any sign of toxicity which may appear later. Any toxicity signs associated with pores and skin, hair, mucus membrane and eyes, food and intake of water, bodyweight, rate of respiration and behavioural, neurological, and autonomic profiles were cited throughout the test duration as reported earlier^14,15.

 

Antitumor Study (ATS):

DAL cells were received from ACI, Thrissur, Kerala, India. The cells inoculateded into SAM (2 × 10⁶cells/ mouse) by intraperitoneal route (i.p). Ascitic fluid was withdrawn from the SAM bearing DAL Tm (on 12^th day of Tm) induction (at the log phase) and suspension was made with phosphate buffer saline (PBS) containing 2 × 10⁶ cells.

 

Treatment schedule:

Sixty male SAM were allocated into six sets or groups (n=10) and extended access to food and water in sufficient quantity. All the SAM received DAL cells in every single set except G-I (2 × 10⁶ cells/ mouse, i.p). G-I considered as normal control (Distilled water p. o.) and G-II was DAL control. After 9^th day of transplantation of DAL, G-III, G-IV and G-V administered with SAS at the dose of 100, 200 and 400mg/kg, p.o for 14 successive days, correspondingly. G-VI administered with benchmark drug 5-fluorouracil (20 mg/kg, p.o) for 14 days continuously¹⁶. After the 14^th day drug administration 5 SAM in every group were sacrificed to evaluate the parameters of growth of Tm such as mean time of survival, cell viability, non-live cell, volume & weight of Tm, and PCV of Tm cell, hematological & biochemical limits and rest of the SAM were maintained in laboratory to find out percentage increase in life expectancy of the Tm host^17,18.

Cytotoxicity Study- In vitro-method:

DAL cells (1X10⁶) in PBS and various conc. (50, 100, 200, 400, 600, 800, 1000, 1600μg/ml) of SAS was stored at 37°C for 3 hrs in 5% CO₂ saturated cell culture flask with filtered cap in a CO₂ incubator¹⁹. The live cells were examined by TBD exclusion method and viability of cell was presented as percentage of control (100 %)²⁰.

 

Parameters of Growth of Tumor (Tm):

Tm Volume and Weight:

14 days after the administration of drug, SAM were sacrificed with excess anesthesia and the from peritoneal cavity, ascetic fluid was taken out. After taking it in centrifuge the volume was measured, and it weighed straight away²¹.

                                  Number of Dead cells

% Cell viability = -------------------------------------- X 100

         Number of viable cells + Number of dead cells

 

Count of Viable and Non-Viable Tm Cells:

TBD exclusion assay was used to observe the live and dead cells. TBD (0.4% in N.S.) was used to stain the cells. The dye is dynamically pumped out from viable cells by mechanism of efflux, whereas non-viable cells were unable to behave in same fashion²². The numbers of live and dead cells were demonstrated by the below mentioned formula:

                (Number of cells × Dilution factor)

Cell count = ------------------------------------------

                  (Area × Thickness of liquid film)

 

Tm PCV:

At 3000rpm for 1 hr, the ascetic fluid was collected. In terms of percentage the volumes of packed cells were demonstrated²³.

 

% Increase in Life Span (ILS):

The effect of SAS on % ILS was observed based on death rate of experimental SAM²⁴.

MST in days = (Day of first death + day of last death)/2.

 

ILS (%) = [(MST of the treated group/MST of the control group)-1] ×100

 

Blood Parameters:

Blood was collected from retroorbital plexus at the last of experiment and estimation of Hb, RBC and WBC count, PCV and DLC was done as per Standard protocol^25,26.

 

Biochemical Parameters:

The remaining portion of blood was used to find out parameters of liver such as Serum glutamic pyruvic transaminase (SGPT), Serum glutamic oxaloacetic transaminase (SGOT), Gamma- glutamyl transpeptidase (GGTP), and alkaline phosphatase²⁷.

Effect of SAS on Solid Tm:

SAM were grouped into three sets (n=5). DAL cells (1×10⁶ cells/mice) were administered intramuscularly (im.) into right hind limb (thigh) of all SAM. The Group I served as DAL Tm control. Group II and Group III treated with SAS 200 and 400mg/kg/p.o for 14 days. Mass of Tm was measured from 15^th day of induction of Tm. Every 5^th day the measurement was carried out for a 30-day period. The formula V= 4/3 πr² used to find out the volume of Tm mass: r=mean; r₁ and r_2: two independent radii of the mass of Tm^28,29.

 

Statistical Analysis:

Data were indicated as mean±S.E.M from 3 independent experimental repeats. Statistical significance (P) was calculated by ANOVA afterwards Dunnett’s test. P values <0.05  were regard as statistically significant.

 

RESULTS:

This study was performed evaluate the ATA of SA which is a herbo-mineral formulation of Indian traditional system of medicine – Siddha, using Dalton's Ascitic Lymphoma in Mice. SA is a combination of total nine ingredients like Dryopteris filix-max, Elemental Mercury (purified), Aconitum napellus, Elemental sulphur (purified), Zingiber officinale, Piper nigrum, Piper longum, Arsenic disulphide (purified), Borax (purified). The composition of SA has been provided in the Table 1.

 

Table 1: The composition of Sivanar Amirtham (SA)

Ingredients	Percentage (%)
Dryopteris filix-max	6.25 %
Elemental Mercury (Purified)	6.25 %
Aconitum napellus.Rt	6.25 %
Elemental Sulphur (Purified)	6.25 %
Zingiber officinale. Rz.	6.25 %
Piper nigrum. Dr.Fr.	50 %
Piper longum. Dr.Fr.	6.25 %
Arsenic disulphide (Purified)	6.25 %
Borax (Purified)	6.25 %

 

Assessment of the acute toxicity (AT) of Sivanar Amirtham (SA):

The AT effect of SAS was evaluated as per the OECD guideline 423, where 2000mg/kg, p.o. dose was used as limit test. No treatment related toxic symptoms or mortality were noticed after oral administration of SAS at a dose of 2000mg/kg. No predominant changes in the gain of body weight were observed and all animals were alive until the finish the experiment suggesting that the median lethal dose (LD₅₀) of SAS is higher than 2000 mg/kg, p.o. The observed parameters were assessed with control group.

 

Antitumor Activity (ATA) of Sivanar Amirtham (SA):

In vitro-cytotoxicity study:

The in vitro cytotoxicity action of SAS was evaluated at various concentrations levels 50, 100, 200, 400, 600, 800, 1000, 1600μg/ml on DAL cells using exclusion method of TBD. The % inhibition of viability of cell was observed as 15, 23, 37, 41, 45, 57, 70 and 84% respectively. The IC₅₀ value was assessed to be 800 μg/ml. The DAL Tm cells stained with TBD showed marked cytolytic activity of SAS 50-1600μg/ml compared to the respective Tm control cells.

 

Effect of SAS on mean survival time (MST):

The effect of SAS on MST is computed in Tab. 2.

The SAS administration by oral route to the Tm induced DAL mice, the MST of DAL control group was observed to be 11.8 d while it increased to 14.8 d and 17.0 d at SAS 100, 200 and 400mg/kg, respectively. Whereas the benchmark drug 5-flurouracil (20mg/kg) treated group showed MST 20.5 d.

 

Effect of SAS on growth of Tm:

SAS treatment (100, 200 and 400mg/kg) predominantly (P<0.05) reduced the volume of Tm, count of viable Tm cells and PCV of Tm cells in a dose reliant approach as compared to that of the DAL control group (Table 2). Furthermore, count of non-viable cells at different doses of SAS were predominantly (P<0.01) increased in a dose dependent manner.

 

The effects of SAS on TG response have been presented in Tab. 2.

 

Effect of SAS on haematological parameters:

Haemoglobin concentration and count of RBC in the DAL control group was decreased as compared to the normal control group with statistical significance (P<0.05). Treatment with SAS at doses 100, 200 and 400mg/kg (p.o.) caused predominant (P<0.01) increase in the above parameters and brought up to the normal levels. The total counts of WBC and PCV was observed to be raised markedly in the DAL control group as compared with the normal group. SAS administration in DAL-bearing SAM significantly reduced the above parameters as compared with the DAL group. In a differential count of WBC, the count of neutrophils (NP) was increased while the lymphocyte (LC), eosinophils (EP) and monocytes (MC) counts were decreased in the DAL group mice. The parameters were stored to normal value after the treatment of SAS as depicted in Table 3.

 

Effect of SAS on biochemical parameters:

The action of SAS on biochemical parameters is shown in Table 4. ALP, SGOT, SGPT, Albumin, Globulin and GGT were enhanced in DAL control group as compared to the normal control group. After treatment with SAS at the dose of 100, 200 and 400mg/kg and 5-FU caused marked decrease in the elevated levels of ALP, SGOT, SGPT, Albumin, Globulin and GGT to normal levels and increased total protein levels.

 

Table 2: Effect of Sivanar Amirtham (SA) on TG parameters

Parameters	DAL	DAL + SAS 100 mg/kg	DAL + SAS 200 mg/kg	DAL + SAS 400 mg/kg	DAL+ 5-FU 20 mg/kg
Mean survival time (d)	11.8±0.37	14.8 ± 0.58*	15.7± 0.58**	17± 0.70**	20.5 ± 0.83**
Increased life span (%)	----------	33.3	37.5	41	62.5
Tm  volume (ml)	13.2  ± 1.06	8.0  ± 1.61**	4.2  ± 0.86**	3.8  ± 0.96**	3.4 ± 0.50**
Tm  PCV (ml)	49  ± 2.53	43.4  ± 1.69ns	39.8 ± 1.35**	34.6  ± 1.36**	31.4  ± 2.24**
Viable cell count (x107 cells/ml)	20 ± 1.22	14.8 ± 1.28*	11.6 ± 1.07**	13.8 ± 1.15**	11 ± 0.70**
Nonviable cell count (x107 cells/ml)	5.4 ± 0.50	9.8 ± 1.068ns	23.4 ± 1.86**	59.2 ± 2.781**	47.8 ± 1.29**

Data are expressed as the mean ± SEM. All groups are compared with DAL control group,** P<0.01, * P<0.05.

 

Table 3: Effect of SAS on hematological parameters

Parameters	Control	DAL	DAL + SAS 100 (mg/kg)	DAL + SAS 200 (mg/kg)	DAL + SAS 400 (mg/kg)	DAL + 5-FU 20mg/kg
RBC (X106/mm3)	5.64 ± 0.37	3.62 ± 0.25ns	5.2 ± 0.15**	5.82 ± 0.29**	6.26 ± 0.27**	5.88 ± 0.31**
HB (g/dL)	15.36 ± 0.53	10.4± 0.50**	12.02±1.31ns	14.82± 0.60**	15 ± 0.31**	14.22 ± 0.67**
Leukocyte (X103/mm3)	8.6 ± 0. 32	10.76± 0.97*	6.98± 0.78**	5.24 ± 0.51**	4.36± 0.24**	4.08 ± 0.30**
Platelets (105/mm3)	4.18 ± 0.10	1.74± 0.16**	4.06± 0.24**	4.3 ± 0.20**	4.23± 0.29**	4.12 ± 0.28ns
MCV (gl)	70.85 ± 3.69	137.24±9.95**	78.39±6.00**	69.28 ± 7.76ns	57.02±7.36**	66.75 ± 11.62**
Neutrophil (%)	40.2 ± 2.65	58.8 ± 3.20*	53.8 ± 5.80ns	47.6 ± 5.69ns	43.4 ± 6.47ns	45 ± 8.15ns
Lymphocyte (%)	54.8 ± 2.31	40.4 ± 2.71ns	46.4 ± 6.73ns	51.2 ± 7.41ns	55.2 ± 8.40ns	51.4 ± 9.42ns
Monocyte (%)	3.0 ± 0.31	2.0 ± 0.31ns	2.6 ± 0.24ns	3.2 ± 0.20ns	4.2 ± 0.73*	5.2 ± 0.58**
Eosinophil (%)	4.0 ± 0.44	1.72±0.14*	4.0 ± 0.54**	5.8 ± 0.37**	5.4 ± 1.10**	5.0 ± 0.70**
Basophil (%)	00 ± 00	00 ± 00	00 ± 00	00 ± 00	00 ± 00	00 ± 00
PCV (%)	35.54 ± 2.04	46.4 ± 1.56*	40.8 ± 2.58ns	39.5 ± 2.36ns	43.69 ± 2.72ns	38.1 ± 5.52ns
MCHC (g/dl)	43.81 ± 2.94	14.43± 0.90ns	29.17±1.93**	37.92± 1.87**	43.69 ± 2.87**	41.41 ± 7.45**

Values are expressed as mean ± SEM. Control Vs SAS treated groups **P<0.01, *P<0.05 and ^nsP>0.05

Table 4: Effect of SAS on biochemical parameters

Parameters	Control	DAL	DAL + SAS 100 (mg/kg)	DAL + SAS 200 (mg/kg)	DAL + SAS 400 (mg/kg)	DAL + 5-FU 20 mg/kg
Total Bilirubin (mg/dL)	0.62 ± 0.05	0.75 ± 0.06ns	0.72 ± 0.09ns	0.76 ± 0.04ns	0.58 ± 0.04ns	0.60 ± 0.68ns
Bilirubin direct (mg/dL)	0.18 ± 0.04	0.39 ± 0.06ns	0.31 ± 0.06ns	0.38 ± 0.07ns	0.21 ± 0.03ns	0.31 ± 0.05ns
Bilirubin indirect (mg/dL)	0.25 ± 0.04	0.44 ± 0.07ns	0.39 ±0.07ns	0.43 ± 0.07ns	0.31 ± 0.04ns	0.37 ± 0.05ns
ALP (U/L)	72.92±2.84ns	125.2 ±1.63**	94.83±1.11ns	74.52 ± 2.57*	83.34 ±  1.41ns	77.18 ± 2.26ns
SGOT (U/L)	65.6 ± 1.44	133.4 ± 1.35*	84 ± 1.40ns	66.2 ± 1.63ns	72.2 ± 2.50ns	66.8 ± 12.03ns
SGPT (U/L)	33.2 ± 1.88	59.4 ± 1.76**	40.4± 3.01*	37.2 ± 2.35*	38.78 ± 8.43*	39.4 ± 4.50*
Total Protein (g/dL)	5.72 ± 0.54	2.68 ± 0.18**	4.96± 0.66**	6.78 ± 0.23**	7.38 ± 0.25**	7.5 ± 0.23**
Albumin (g/dL)	2.94 ± 0.31	5.56 ± 0.25**	5.76 ±0.89ns	3.32 ± 0.16*	3.38 ± 0.12*	2.92 ± 0.36ns
Globulin (g/dL)	3.4 ± 0.42	5.22 ± 0.26**	3.88±0.22**	3.22 ± 0.14**	2.86 ± 0.26**	3.06 ± 0.30**
GGT (U/L)	27.4 ± 3.09	45.2 ± 3.51**	34.8 ±2.17ns	31.4 ± 2.40*	30.2 ± 3.58**	28.6 ± 2.67**

Data are expressed as the mean ± SEM. Control groups are compared with DAL group and DAL groups are compared with SAS treated groups **P<0.01, *P<0.05 and ^nsP>0.05.

 

Table 5: Effect of Sivanar Amirtham (SA) on solid growth of tumor (Tm)

Solid Tm volume in ml
Groups	15th day	20th day	25th  day	30th day
DAL control	26.03 ± 1.38	28.16 ± 1.48	29.32 ± 1.45	30.69 ± 0.62**
SAS 200 mg/kg	21.67 ± 1.40ns	17.26 ± 1.82**	13.13 ± 1.54**	12.94 ± 0.07**
SAS 400 mg/kg	14.70 ± 2.74**	11.93 ± 1.83**	8.49 ± 0.67**	8.30± 0.19**

 

Effect of SAS on solid growth of Tm:

Nowadays the traditional systems like Siddha, Unani etc., are much more in use and effective for the treatment of various diseases³⁰. There was a reduction in the volume of Tm of SAM treated with SAS at 200 and 400mg/kg from 2 to 4 weeks. At 4-week, volume of Tm of DAL control SAM was 30.69ml, (P<0.01) whereas for the SAS treated group (200 and 400mg/kg) it was found to be 12.94 and 8.30ml respectively. The effects of SAS on solid TG response have been presented in Tab. 5.

 

In this study, the AT study following OECD guidelines-423 showed that SAS up to 2000mg/kg are non-toxic and safe. The present research was conducted to evaluate the ATA of SAS in DAL Tm bearing SAM. The SAS treated SAM at the doses of 100, 200 and 400 mg/kg p.o predominantly inhibited the volume of Tm, count of viable Tm cell, PCV and reverted the haematological and biochemical parameters to normal levels. Treatment with SAS increased the life span of the Tm bearing mice^31,32.

 

It was exhibited that the presence of Tm in the human or in the experimental SAM is known to affect the physiology of liver. The significantly elevated levels of SGOT, SGPT, ALP in serum of Tm inoculated SAM indicated dysfunction of liver and loss of cell membrane integrity. The marked reversal of these modifications towards the normal by SAS treatments³³. In the present work, the biochemical investigation of DAL carrying SAM represented predominant changes representing the lethal effect of the Tm. The normalization of these actions detected in the serum administered with SAS possesses significant ATA of the drug.

 

DISCUSSION:

The present work was conducted out to estimate the ATA of SA in DAL bearing SAM.The SAS administered to SAM at the doses 200 and 400mg/kg (p.o.) markedly repressed the volume of Tm, PCV of Tm cell, count of Tm cell and modified the haematological factors to closely to the standard levels. In DAL carrying SAM an everyday speedy growth in volume of ascites of Tm was noted. Ascites fluid is linked with rapid growth of Tm cells as act as a direct nutritive source for Tm. Treatment with SAS augmented the proportion of TBD (+ve) stained non-viable cells in tumour carrying SAM. The trustworthy criteria for assessing the action of any anti-CA drugs are the increase in the life expectancy of SAM³⁴.

 

The SAS dropped the fluid volume of ascites, feasible cell number and increased life expectancy. It may be summarized that SAS by dwindling the volume of nutritive fluid and blocking the excrescence development, this could be the basis for the increment in life expectancy of DAL carrying mice. Generally, in CA chemo treatment the major drawbacks that are being confronted are of myelosuppression & anaemia. The anaemia met in excrescence bearing SAM is substantially due to decrease in RBC or haemoglobin chance and this may be because of insufficiency of iron or due to haemolytic or myelopathic ailments^35,36.

 

In the hematologic parameter, administration of SAS at doses 100, 200 and 400 mg/kg (p.o.) produced important increase within the haemoglobin content and blood cell count and mentioned to the common levels. Treatment with SAS in DAL carrying SAM considerably decreased the WBC count and conjointly reduced the PCV with respect to the DAL group. In an exceedingly differential count of WBC, the presence of NP was increased whereas the LC, EP and MC counts were reduced within the DAL group mice³⁷.

 

After treatment with SAS at the dose of 100, 200 and 400 mg/kg and 5-FU caused important decrease within the elevated levels of ALP, SGOT, SGPT, Albumin, Globulin and GGT to near normal levels and raised the levels of total protein³⁸.

 

After administration of SAS at the dose of 200 and 400 mg/kg solid growth volume of SAM was reduced from two to four weeks. All these information confirmed that the SAS are often used as a possible agent within the space of CA therapy³⁹. The investigations need to be administered in characterization and therefore the mechanism involving in growth and cytotoxic impact. Hence, it is often complete that the SAS has the tumoricidal impact comparable normal drug 5-FU effectivity and thereby maintain traditional physiological profile in mice^40,41.

 

CONCLUSION:

The current investigation demonstrates that the traditional medicine, SAS may be considered as a relatively safe at doses consumed. It did not cause either toxic symptoms or mortality of rodents during the acute periods of study. The SAS was extensively investigated for its ATA against DAL induced Tm in mice. The SAS at different doses showed predominant ATA with respect to DAL control SAM. Thus, the results of the current work support the conventional and clinical claims of the SAS in the therapy of Tm.

 

CONFLICT OF INTEREST:

The authors declare no conflicts of interests.

 

ACKNOWLEDGEMENT:

Authors are grateful to Dr. V. Madhavan, Dean, Faculty of Pharmacy, M.S.R.U.A.S., Bangalore, for providing all the facilities to carry out this research work. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

 

REFERENCES:

1.      Armour FE, Blood FR, Belden DA. The manual for laboratory work in mammalian Physiology. The University of Chicago Press Chicago. 1965; 3: 4-6.

2.      Alkhatib MH, Alshehri WS, Abdu FB. In vivo evaluation of the anticancer activity of the gemcitabine and doxorubicin combined in a nanoemulsion. J Pharm Bioall Sci. 2018; 10(1): 35-42. doi: https://doi.org/10.4103/jpbs.JPBS_225_17

3.      Baidya M, Anbu J, Akhtar MS, Sarkar S, Mandal SK. Antimicrobial Evaluation of Ethanolic Extract of Selected Seed Shells. Int J Curr Pharma Res. 2020; 12(6): 74-76. doi: https://doi.org/10.22159/ijcpr.2020v12i6.40286.

4.      Bala A, Biswakanth K, Haldar PK, Mazumder UK, Bera S. Evaluation of anticancer activity of Cleome gynandra on Ehrlich’s Ascites Carcinoma treated mice, J. Ethnopharmcol. 2010; 129(1): 131-134. doi: https://doi.org/10.1016/j.jep.2010.03.010.

5.      Coussens L, Werb Z. Inflammation and cancer. Nature. 2002; 420: 860–867. doi:https://doi.org/10.1038/nature01322.

6.      Mandal SK, Biswas D, Sony A, De A, Dastider D, Baidya M, Paul S, Dawn S, Mandal S, Plants and phytochemicals in the treatment of colorectal Cancer: recent updates, Int. J. Biol. Pharm. Allied Sci. 2021; 10(10): 3502-3521. doi:org/10.31032/IJBPAS/2021/10.10.5657.

7.      Clarkson BD, Burchenal JH. Preliminary screening of antineoplastic drugs. Prog. Clin. Cancer. 1965; 1: 625-629.

8.      Dacie JV, Lewis SM. Practical Haematology. (12th edition)  Elsevier, 2017; doi: https://doi.org/10.1111/bjh.14872.

9.      Dahab RA, Afifi F. Antiproliferative activity of selected medicinal plants of Jordan against a breast adenocarcinoma cell lines (MCF7). Sci Pharm Inc. 2007; 75(3): 121-136. doi:https://doi.org/10.3797/scipharm.2007.75.121

10.   Farah AO, Nooraain H, Noriham A, Azizah AH, Nurul-Husna R. Acute and oral subacute toxicity study of ethanolic extract of Cosmos caudatus leaf in Sprague Dawley rats. Int J Biosci Biochem Bioinforma. 2013; 3(4): 301–305. doi:https://doi.org/10.7763/IJBBB.2013.V3.218

11.   Graham JG, Quinn M.L, Fabricant DS, Farnsworth NR. Plants used against cancer-an extension of the work of Jonathan Hartwell. Journal of Ethnopharmacol. 2000; 73: 347-377. doi:https://doi.org/10.1016/S0378-8741(00)00341-X.

12.   Ganapathy P, Elumalai K, Arumugam MK, Amulya CS, Manivel R. Anticancer potential of Siddha formulations against oral cancer cell line in vitro. Trends Med. 2019; 19: 1-6. doi:https://doi.org/10.15761/TiM.1000192.

13.   https://www.ism.kerala.gov.in. Accessed on 06/12/2021.

14.   https://www.ayurmedinfo.com › 2019/11/25 › sivanar-amirtham. Accessed on 06/12/21.

15.   Hogland HC. Hematological complications of cancer chemotherapy. Semin Oncol . 1982; 9: 95-102.

16.   Muthu IS, Kanth SB, K. Kalimuthu K, Sangiliyandi G. Antitumor activity of silver nano particles in Dalton’s lymphoma ascites tumor mode.  Int. J. of Nano Med. 2010; 5: 753-762. doi:https://doi.org/10.2147/IJN.S11727

17.   Baidya M, Jayaraman A,  Maji HS, Ramya Krishna PS, Das D, Evaluation of acute and sub-acute toxicity of sivanar amirtham in albino mice and wistar albino rats, J. Med. P’ceutical Allied Sci. 2022; 11(1): 4196 - 4204. doi: 10.22270/jmpas. V11I1.2217.

18.   Venu Gopal Y, Ravindranath A, Kalpana G, Rajkapoor B, Sreenivas SA . Antitumor and antioxidant activity of Diospyros peregrina against Dalton’s ascites lymphoma in rodents. Annals of Biological Research. 2012; 3 (11):4985-4992.

19.   Ramya Krishna PS, Jayaraman A, Baidya M, Nayak DA.Toxicological evaluation of fucoidan, a polysaccharide isolated from Turbinaria conoides (J. agardh) Kutzing procured from mandapam coastal area, tamilnadu, J. Med. P’ceutical Allied Sci. 2022; 11(2): 4629 – 4635. doi: 10.55522/jmpas.V11I2.2511.

20.   Gupta M. Antitumor Activity and Antioxidant Status of Caesalpinia bonducella against Ehrlich Ascites Carcinoma in swiss albino mice. J. Pharmacol. Sci. 2004; 94(2): 177-184. doi:https://doi.org/10.1254/jphs.94.177.

21.   Price VE, Greenfield RE. Anemia in cancer. Adv. Can. Res. 1958; 5: 199-200. doi:https://doi.org/10.1016/s0065-230x(08)60413-3. 

22.   Babu P, Anbu J, Ashwini A, Velpandian V. Anti tumour activity of guru pathangam against dalton ascites lymphoma (dal) in mice. Int J Pharm Bio Sci. 2012; 3(3): 281 – 289.

23.   Meera R, Chidambaranathan N. Anti cancer Activity of Ethanolic Extract of Crataeva magna Lour (DC) against Ehrlich Ascitic Carcinoma Cell Lines in Mice. J. Pharm. Sci. and Res. 2017; 9(10): 1869-1873.

24.   Baidya M, Maji HS, Bhatt S, Das D, In-vitro Evaluation of the Thrombolytic and Anti-inflammatory Activity of Capparis sepiaria Root Extracts, J. Med. P’ceutical Allied Sci. 2022; 11(1), 4166-4171. doi: 10.22270/jmpas.V11I1.1879.

25.   Rajesh kumar NV, Kuttan G, Ramsewak RS, Nair MG, Kuttan R. Antitumour and anticarcinogenic activity of Phyllanthus amarus extract. J. Ethnopharmacol.2002; 81(1): 17-22. doi:https://doi.org/ 10.1016/s0378-8741(01)00419-6.

26.   Byahatti S, Bogar C, Bhat K, Dandagi G.Evaluation of anticancer activity of Melaleuca alternifolia. (i. e. tea tree oil) on Leukemia cancer cell line (K562): An in vitro study. J. Med. Plants Stud. 2018; 6(5): 01-06.

27.   Sheeja KR, Kuttan G, Kuttan R. Cytotoxic and antitumour activity of Berberin. Amala Res. Bull.1997; 17:73-76.

28.   Sur P, Ganguly DK. Tea plant roots extract (TRE) as an antineoplastic agent. Planta Med. 1994; l60: 106-109. doi:https://doi.org/10.1055/s-2006-959427.

29.   Yadavalli V.Evaluation of anticancer compounds from suspension cultures of Holy Basil (Ocimum sanctum L.) Int. J. Green Pharm. 2019; 13 (2): 98. https://doi.org/10.22377/IJGP.V13I2.2487

30.   Mandal SK, dawn S, Mondal S, Sarkar S, Mandal R, Baidya M, Paul S, Pramanick A, Dastider D, Sen DJ, Das A, Kolay A, The therapeutic versatility of quinolines, Int. J. Biol. Pharm. Allied Sci. 2021; 10(7): 2161-2181. doi:org/10.31032/IJBPAS/2021/10.7.5528.

31.   Wintrobe MM, Lee GR, Boggs DR. Clinical Hematology .1961; 5, Philadelphia.

32.   Sanakousar K. Patel, Arun K Shutter, Ria Patil, Archana Desangi, Vishalakshi Malali, Jyoti Patil, Sumangala Patil, Kusal K. Das, Prachi P. Parvatikar. In-Vitro Antioxidant, Anti-Inflammatory and Cytotoxic effects of different Solvent Extraction Terminalia chebula, Terminalia billerica, Phyllanthus emblica. Research Journal of Pharmacy and Technology. 2022; 15(7): 2940-4. doi: 10.52711/0974-360X.2022.00490.

33.   A. Mohamed Niyaz, S. Ravikumar. Antilarval and in vitro Anticancer efficacy of Cladode extracts of Opuntia dillenii (Ker Gawl.) Haw., Cereus pterogonus Lem. and Acanthocereus tetragonus (L.) Hummelinck. Research Journal of Pharmacy and Technology. 2022; 15(7): 2877-2. doi: 10.52711/0974-360X.2022.00480.

34.   J. Mariya Jothi, A. Jayaprakash. Phytochemical Analysis and In Vitro Anticancer Activity of Enteromorpha compressa against Ovarian Cancer.Research Journal of Pharmacy and Technology. 2022; 15(5): 1943-7. doi: 10.52711/0974-360X.2022.00323

35.   Yogita S. Ozarde, Vishnu P. Choudhari. Preliminary Qualitative Phytochemical Analysis and Acute Oral Toxicity Study of Latex of an Ethnomedicinal Plant Euphorbia fusiformis Buch.-Ham.Ex D.Don. Research Journal of Pharmacy and Technology. 2022; 15(3): 1123-7. doi: 10.52711/0974-360X.2022.00188.

36.   Hemant Devidas Une, Lalita Bansidas Bhagure. The Anti-leukemic Potential of Cyclea peltata as Validated by Phytochemical and Cell Line Studies. Research Journal of Pharmacy and Technology. 2022; 15(3): 1064-0. doi: 10.52711/0974-360X.2022.00178.

37.   Amaq Fadholly, Arif N. M. Ansori, Budi Utomo. Anticancer Effect of Naringin on Human Colon Cancer (WiDr Cells): In Vitro Study. Research Journal of Pharmacy and Technology. 2022; 15(2): 885-8. doi: 10.52711/0974-360X.2022.00148.

38.   Fitriyanti Jumaetri Sami, Nunuk Hariani Soekamto, Tatsufumi Okino, Firdaus, Jalifah latip. A Flavonoid compound of Turbinaria decurrens Bory with The Potential Antioxidant and Anticancer Activity. Research Journal of Pharmacy and Technology. 2021; 14(12): 6207-0. doi: 10.52711/0974-360X.2021.01074.

39.   Asish Bhaumik, Samaresh Datta, Susmita Datta, Radheshyam Samanta, B. D. Tripathi. Extraction and Isolation of Phenolic compounds from Sweet lime and Evaluation of Anticancer potentiality followed by Molecular docking against Topoisomerase II. Research Journal of Pharmacy and Technology. 2021; 14(11): 5993-7. doi: 10.52711/0974-360X.2021.01041.

40.   Raghad J. Fayyad, Alaa Naseer Mohammed Ali, Noor T. Hamdan. The Specific Anti-cancerous Mechanisms Suggesting Spirulina Alga as a Promising breast Cancer Fighter. Research Journal of Pharmacy and Technology 2021; 14(10): 5599-2. doi: 10.52711/0974-360X.2021.00974

41.   Syafruddin Ilyas, Rostime H. Simanullang, Salomo Hutahaean, Rosidah Rosidah, Putri C. Situmorang. Effect of Zanthoxylum acanthopodium methanol extract on CDK4 expression to cervical cancer. Research Journal of Pharmacy and Technology. 2021; 14(11): 5647-2. doi: 10.52711/0974-360X.2021.00982.

 

 

 

 

 

Accepted on 03.05.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(12):5677-5683.