INTRODUCTION:

Several epidemiologic and animal model studies suggested that dietary or herbal chemicals remove or alleviate cancer by their antioxidant property leading to apoptosis¹. Breast cancer emerged as the second most lethal threat to human beings and the current treatment pattern pose risk in the development of multiple complications. Natural resources promise a safe way of combating cancer symptoms.

Azima tetracantha (Salvadoraceae) is a well known medicinal herb, termed ‘Mulsangu’ in Tamil and 'Kundali' in Sanskrit. Root, root bark and leaves of Azima tetracantha (lam) are used with food as a remedy for rheumatism, diuretic and as stimulant¹. Traditionally Indian medical practitioners use Azima tetracantha (lam) in inflammatory conditions, cough, asthma, small pox and diarrhoea^2,3. The major phyto-constituents reported in Azima tetracantha (lam) are azimine, azecarpin, carpine, isorhamnitine-3-O-rutinoside, friedelin, lupeol, glutinol and β-sitosterol^4,5. Azima tetracantha (lam) is reported to have antifungal⁶antitumour⁷, antidiabetic⁸, antidiarrhoeal⁹ and hepatoprotectiveactivities.

Azima tetracantha (lam) is a low, spinouts, highly branched bush, woody below but with pale green, herbaceous, almost quadrangular young branches. The leaves are in opposite to sub-opposite, decussate pairs. They are shortly petiolate, about 2x4cm long, entire, elliptic, acute, sharply mucronate, rigid, pale green with an acute base. Usually, there are two laterally placed spines in the axil of a leaf. The spines which morphologically represent the first pair of leaves of the auxiliary shoot are about three cm long, more or less, triangular in cross section, very sharp and with an indurate apex. The plant is dioeciously. The flowers are borne in the axils of leaves. Generally, there is cymes of three flowers in the axil of a leaf which is the upper branches, especially of the male plants become greatly reduced or even completely suppressed.

The detection and analyses of phytochemical constituents within medicinal plants extracts have relied on a number of chromatographic and spectrometric techniques such as TLC, UV, IR and NMR¹⁰. Of recent, phytochemical analysis of medicinal plant extracts involve sophisticated techniques that include Gas chromatography instrument coupled with mass spectrometer (GC-MS). GCMS affords direct analysis of the phytochemicals present in medicinal plant extracts¹¹.

Uses:

The plant is used in indigenous medicines for rheumatism, microbial infections, diahorrea, inflammatory conditions, reduce lipid and as hepato-protective.

MATERIALS AND METHODS:

Collection of plants:

The aerial part (leaves) of Azima tetracantha (lam) was collected from the Panayur area of Madurai, Tamilnadu as raw material, during the second week of February 2015 and a voucher specimen is stored in C.L. Baid Mehta College of Pharmacy (001/ATL/CLBP) and the plant material was authenticated by a renowned botanist. About 500 g of coarse powdered leaf in 2.5 L water is boiled, cooled and filtered. The filtrate is evaporated to dryness in desiccator and stored in refrigerator (Yield- 26.5% w/w). The all extracts of Azima tetracantha (lam) was subjected to preliminary phytochemical analysis¹²

Various extraction methods for isolation of constituents:

The whole plant will be subjected to shade drying and extraction with petroleum ether (60-80^oC) chloroform, Ethyl acetate and 80% ethanol in soxhlet apparatus by simultaneous extraction each for 72 hours. Concentrate the solvents in vacuum. The crude solid obtained on evaporation are to be studied for preliminary qualitative phytochemical evaluation.

Isolation:

This extract was concentrated in vacuum and subjected to flash column chromatography over TLC grade silica gel (60-120 mesh). Elution of the column first with petroleum ether, chloroform increasing amounts of EtOAc in petroleum ether and finally with methanol yielded a number of fractions. The proportion of solvent systems used to obtain compound I (10mg) , compound II (15mg) and compound III (14mg) were 85% chloroform: 15% Etoac, 75% choloroform: 25% Etoac, 98% chloroform: 2% MeOH from fractions 4,6 and 8. The compounds were identified by gas chromatography mass spectroscopy as Friedelin, myricetin and isorhamnitine-3-O-rutinoside.

Phytochemical Screening:

The extract was subjected to phytochemical analysis to test the presence of carbohydrates, glycosides, alkaloids, flavonoids, terpenoids, tannins, sterols, and saponins in leaf extracts.

Animals and drugs:

Female Sprague–Dawley rats weighing (220±5 g) were procured from central animal house, C.L. Baid Mehta College of Pharmacy, Chennai, Tamil Nadu, India. The animals were housed in well ventilated large spacious polypropylene cages and had 12±1 hr light and dark cycle throughout the experimental period. The animals received a balanced diet of commercially available pellet rat feed and water ad-libitum. The ethyl acetate and ethanol extract and isolated compounds II and III of Azima tetracantha LAM leaf and tamoxifen dissolved in corn oil and administered intragastrically.

Tumor induction:

Mammary tumor was induced by a single dose of 20 mg of 7, 12-Dimethyl benzanthracene (DMBA) dissolved in corn oil (1 ml) given through an oral gavage¹³. All the experimental animals were sacrificed after 90 days.

Experimental design:

A total of 66 female Sprague–Dawley rats were divided into eleven groups of six rats each. Group I received normal saline (10 ml/kg body weight), Group II tumor control, Group III received standard tamoxifen (10 mg/kg body weight), Group IV received Ethyl acetate extract of Azima tetracantha LAM (200 mg/kg body weight) and tamoxifen (10 mg/kg body weight), Group V received ethanol extract of Azima tetracantha LAM (200 mg/kg body weight) and tamoxifen (10 mg/kg body weight), Group VI and Group VII received ethyl acetate and ethanol extract of 200 mg/kg body weight, respectively. Group VIII received Compound II of Azima tetracantha LAM (200 mg/kg body weight) and tamoxifen (10 mg/kg body weight), Group IX received Compound III of Azima tetracantha LAM (200 mg/kg body weight) and tamoxifen (10 mg/kg body weight), Group X and Group XI received Compound II and Compound III of 200 mg/kg body weight, respectively. At the end of the experimental period, all the rats were alive and were anesthetized with diethyl ether and sacrificed by euthanasia. Animals were starved overnight before sacrifice. Blood was collected and the serum was separated by centrifugation. Breast was dissected out and washed with ice-cold 0.9% NaCl solution. The resultant solid tumor was considered to be prelate ellipsoid with one long axis and two short axes. The two short axes were measured with a vernier caliper. The tumor weight ¹⁴was calculated using the following formula:

Weight= length x width/2

Tissues (100 mg) were homogenized in 0.1 M Tris–HCl buffer (pH 7.4). The homogenate was used for the determination of various antioxidant biochemical parameters.

Biochemical analysis:

The superoxide dismutase (SOD) activity was measured at absorbance 420 nm using a spectrophotometer as the degree of inhibition of autoxidation of pyrogallol in an alkaline pH according to the method of Marklund and Marklund¹⁵. The catalase (CAT) activity was assayed by the method of Sinha¹⁶. The activity of glutathione peroxidase (GPx) was assayed by the method of Rotruck et al.¹⁷. Reduced glutathione (GSH) was determined by the method of Moron et al.¹⁸. Vitamin E (a-tocopherol) levels were estimated by the method of Desai¹⁹. Vitamin C (ascorbic acid) was measured by the method of Omaye et al.²⁰. Lipid peroxidation was estimated by the method of Högberg et al.²¹

Western blot analysis:

Tumors were disaggregated by treatment with an enzymic mixture containing 2.0 g/l collagenase, 0.5 g/l proteases, and 2.0 g/l DNase for 90 minutes at 37°C. The resulting cell suspensions were filtered through a 30 μm nylon mesh. Centrifuged cells were washed in phosphate buffered saline and boiled in Laemmli lysis buffer for 5 minutes. Breast tissue proteins (50 μg/lane) were separated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane. The membrane was probed with B-cell lymphoma-2 (Bcl-2) specific antibody (1:2000 dilution) to determine their levels, and the intensities were measured using Image J/Fiji 1.46. β-actin was used as an internal control²².

Statistical analysis:

The treated groups were compared with the toxicant control groups. All results were expressed as a mean ±standard error of the mean of six animals in each group. The results were analyzed statistically using one-way ANOVA followed by Newman-Keuls multiple comparison methods to compare the mean value of different groups using Graph pad 3.1 version. p<0.05 was considered significant.

RESULTS:

The ethyl acetate and ethanol extract of Azima tetracantha LAM leaf screened for phyto constituent analysis revealed the presence of alkaloids, phenols, flavonoids, amino acids, quinones, steroids, and carbohydrate in various colour reactions.

Table 1 presents the body weight, tumor weight and thio barbituric acid-reactant substances of control and experimental animals. The body weight was found to be significantly decreased in Group II tumor induced animals when compared with control animals (p<0.01). Conversely, the administration of Ethyl acetate and ethanol extract increased the body weight in Group III to XI when compared to Group II animals (p<0.01). The Groups III to IV animals exhibited significant (p<0.01) reduction of tumor weight when compared to Group II. The Groups V to VI animals exhibited significant (p<0.05) reduction of tumor weight when compared to Group II. The Groups VII to XI animals exhibited significant (p<0.05) reduction of tumor weight when compared to Group II. Administration of Ethyl acetate and ethanol extract and isolated compounds II and III decreased the tumor weight significantly (p<0.05).

Activities of enzymic and non-enzymic antioxidants in breast homogenate and serum of control and experimental animals are presented in Tables 2 and 3, respectively. Group II cancer-bearing animals showed a significant reduction in both enzymic and non enzymic antioxidant levels (p<0.01) when compared to control animals. Administration of tamoxifen and Ethyl acetate and ethanol extract in Groups III to VI animals significantly (p<0.01) increased the antioxidant levels when compared to Group II animals. Striking results were observed for body weight, enzymic and non-enzymic parameters for Group IV, by which the combined efficacy of standard and Ethyl acetate and ethanol extract at low dose is understood. To assess the expression of Bcl-2 in breast tissues of experimental animals, SDS-PAGE and Western blot analysis were performed. Intensities observed of treated and normal groups were significantly different from Group II. Group IV intensity demonstrated an excellent silencing of Bcl-2 expression in combined treatment pattern of standard and extract even with Ethyl acetate and ethanol extract, isolated compounds II and III treated alone.

DISCUSSION:

The present study indicates that administration of plant extract resulted in substantial inhibition of breast tumor incidence or decrease in the initiation of tumor genesis and increase in body weight of animals treated with Ethyl acetate and ethanol extract , isolated compounds II and III at a dose of 200 mg/kg and 400 mg/kg body weight. Toxic manifestation of DMBA is associated with its oxidative metabolism leading to the formation of reactive metabolites (epoxides and quinines) capable of generating free radicals. Metabolism of DMBA by the mixed function oxidases system often results in the formation of oxy radicals which bind covalently to nucleophillic sites on cellular macromolecules thereby eliciting cancerous responses²³. The generation of reactive oxygen species (ROS) and the peroxidation of membrane lipids are well associated with the initiation of carcinogenesis affecting the normal biochemical process, which further leads to the reduction of body weight²⁴. Naturally, there is a dynamic balance between the amount of free radicals generated in the body and antioxidant defense system that quench or scavenge them and thereby protect the body against pathogenesis²⁵. It is evident from the results that increased the level of lipid peroxides (LPO) was found in cancer-bearing animals when compared to control group. On the contrary, reduced level of LPO was observed in tamoxifen and Ethyl acetate and ethanol extract, isolated compounds II and III treated animals at both doses indicating that it is a good free radical scavenger. SOD and CAT act mutually supportive anti oxidative enzymes, which provide protective defense against ROS²⁶. The present study reveals that SOD levels are decreased in the cancer-bearing animal, which may be due to altered antioxidant status caused by carcinogenesis. The present study also shows that decreased level of CAT observed in Group II cancer-bearing animals may be due to the utilization of antioxidant enzymes in the removal of H₂O₂ by DMBA.

GPx is an important defense enzyme against oxidative damage and this, in turn, requires GHS as a cofactor. GPx catalyses the oxidation of GSH using H₂O₂²⁷. Our findings agree well with this observation and also the activity of GPx significantly decreased in cancer-bearing animals.

The non-enzymic antioxidant systems are the second line of defense against free radical damage. GSH is an important non-protein cellular thiol that in conjunction with GP_x plays a regulatory role in cell proliferation²⁸. We observed decreased activity of GSH in cancer bearing animals. The Ethyl acetate and ethanol extract, isolated compounds II and III increased the GSH levels, which clearly suggest their antioxidant property. Decreased levels of water soluble antioxidants found in cancer-bearing animal may be due to the utilization of antioxidants to scavenge the free radicals.

Vitamin E is a potent oxygen radical scavenger that protects cell membranes from oxidative damage initiated by carcinogens²⁹. The present study found decreased levels of vitamin E in the cancer condition which may be due to excessive production of ROS by cancer cells. The free radical clearing capacity of vitamin E is due to the localization of an unpaired electron on its conjugated double bond.

The apoptotic signal transduction pathway commonly induced by anticancer agents is associated with the induction of Bax and cleaved PARP and the down regulation of Bcl-2 and pAkt. Bcl-2 expression is bcl-2 gene³⁰, such that over expression of Bcl-2 might be expected to confer greater drug resistance on ER-positive breast cancer cells. Some reports suggest that Bcl-xL, but not Bcl-2, is capable of modulating apoptosis induced by tumor necrosis factor related apoptosis ligand.

Usually, the down regulation of Bcl-2 expression by AS Bcl-2 enhances drug sensitivity by modulating the apoptotic signal transduction pathway of Bcl-2³¹. However, Bcl-2 inhibits Bid-induced apoptosis at the mitochondrial level by blocking cytochome c release, whereas Bcl-Xl does not affect the insertion of tBid into mitochondrial membranes^32. So in this study, Bcl-2 is concentrated rather than any other member of the same family. Over expression of Bcl-2 is observed more frequently than over expression of Bcl-xL (70% vs. 40%) in breast cancer tissue, which suggests a more important role for Bcl-2 in conferring drug resistance. In our results greater suppression of Bcl-2 was achieved.

Table 1: Effect of on body weight and tumor weight of control and experimental Animals

GROUPS	BODY WEIGHT(g)	TUMOUR WEIGHT(g)	BREAST TBARS (mM/mg tissue)
I	220.30±9.25	-	0.28±0.03
II	145.55±5.85*a	6.15±0.32	0.09±0.01*b
III	185.40±6.08*b	2.65±0.24*b	0.24±0.02*b
IV	198.30±6.15*b	2.40±0.20*b	0.26±0.03*b
V	189.70±6.25*b	3.30±0.28*b	0.19±0.02*b
VI	195.40±6.40*b	3.05±0.24*b	0.22±0.02*b
VII	193.25±5.26*b	3.01±0.21*b	0.18±0.01*b
VIII	194.38±2.57*b	2.99±0.20*b	0.21±0.03*b
IX	190.65±3.86*b	3.03±0.21*b	0.20±0.01*b
X	191.23±3.43*b	3.00±0.28*b	0.21±0.01*b
XI	192.41±2.21*b	3.07±0.18*b	0.19±0.03*b

Values are expressed as mean ± SEM. All values were find out by using one-way ANOVA followed by Newman-Kevls multiple range tests. *a Values are significantly different from normal control at p <0.01. *b Values are significantly different from breast cancer control at p <0.01. TBARS: Thiobarbituric acid reactive substances, SEM: Standard error of mean.

Table 2: Effect on enzyme and non-enzyme antioxidants in breast homogenate for control and experimental animals

GROUPS	SOD	CAT	GP_X	GSH	VIT C	VIT E
I	16.35±1.70	69.15±1.88	12.85±0.80	13.12±0.96	3.60±0.35	5.32±0.53
II	7.78±0.65*a	43.60±1.32*a	6.92±0.65*a	6.28±0.71*a	1.58±0.20*a	3.42±0.26*a
III	15.28±0.86*b	68.64±1.74*b	12.60±0.72*b	12.88±0.80*b	3.36±0.30*b	5.18±0.46*b
IV	15.66±0.92*b	68.78±1.76*b	12.71±0.76*b	12.96±0.86*b	3.44±0.32*b	5.24±0.48*b
V	15.15±0.80*b	67.95±1.48*b	11.95±0.68*b	12.12±0.66*b	3.15±0.24*b	4.96±0.36*b
VI	15.48±0.88*b	68.18±1.56*b	12.42±0.74*b	12.62±0.78*b	3.30±0.28*b	5.05±0.38*b
VII	15.34±0.82*b	67.25±1.32*b	12.39±0.71*b	12.37±0.75*b	3.27±0.25*b	5.03±0.32*b
VIII	15.21±0.85*b	68.76±1.44*b	12.25±0.53*b	12.23±0.71*b	3.29±0.29*b	5.01±0.39*b
IX	15.68±0.87*b	66.97±1.85*b	12.63±0.21*b	12.59±0.76*b	3.28±0.24*b	5.06±0.35*b
X	15.49±0.86*b	67.34±1.21*b	12.15±0.86*b	12.65±0.72*b	3.30±0.26*b	5.02±0.36*b
XI	15.91±0.81*b	68.21±1.28*b	12.21±0.72*b	12.60±0.78*b	3.31±0.27*b	5.05±0.31*b

Values are expressed as mean±SEM. All values were find out by using one-way ANOVA followed by Newman-Kevls multiple range tests. *aValues are significantly different from normal control at p <0.01. *bValues are significantly different from breast cancer control at p <0.01. SEM: Standard error of mean, SOD: Superoxide dismutase, CAT: Catalase, GPx: Glutathione peroxidase, GSH: Reduced glutathione.

Table 3: Effect of enzyme and non-enzyme antioxidants in serum for control and experimental animals

GROUPS	SOD	CAT	GP_X	GSH	VIT C	VIT E
I	12.10±1.40	63.22±3.50	9.68±1.20	10.82±1.08	2.95±0.08	5.08±0.48
II	9.05±0.92*a	34.60±2.45*a	6.86±0.88*a	7.22±0.72*a	1.58±0.06*a	3.36±0.30*a
III	11.76±1.08*b	57.45±3.12*b	9.12±1.02*b	10.18±0.92*b	2.68±0.07*b	4.48±0.36*b
IV	11.89±1.18*b	59.20±3.20*b	9.18±1.08*b	10.44±0.98*b	2.79±0.08*b	4.54±0.40*b
V	11.26±1.05*b	51.66±2.96*b	8.44±0.96*b	9.30±0.82*b	2.26±0.06*b	4.16±0.31*b
VI	11.56±1.14*b	54.34±3.08*b	8.92±1.0*b	9.86±0.94*b	2.48±0.07*b	4.32±0.32*b
VII	11.52±1.12*b	54.31±3.03*b	8.89±1.0*b	9.84±0.91*b	2.42±0.06*b	4.36±0.36*b
VIII	11.54±1.11*b	54.30±3.01*b	8.91±1.0*b	9.81±0.96*b	2.45±0.05*b	4.33±0.32*b
IX	11.58±1.13*b	54.29±3.05*b	8.90±1.0*b	9.85±0.99*b	2.49±0.02*b	4.35±0.31*b
X	11.59±1.14*b	54.28±3.02*b	8.93±1.0*b	9.80±0.90*b	2.41±0.06*b	4.31±0.30*b
XI	11.51±1.10*b	54.33±3.05*b	8.90±1.0*b	9.88±0.92*b	2.46±0.08*b	4.34±0.31*b

Values are expressed as mean±SEM. All values were find out by using one-way ANOVA followed by Newman Kevls multiple range tests. *aValues are significantly different from normal control at p <0.01. *bValues are significantly different from breast cancer control at p <0.01.SEM: Standard error of mean, SOD: Superoxide dismutase, CAT: Catalase, GPx: Glutathione peroxidase, GSH: Reduced glutathione.

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Received on 16.10.2018 Modified on 21.11.2018

Res. J. Pharmacology and Pharmacodynamics.2019; 11(1): 05-10.

DOI: 10.5958/2321-5836.2019.00002.8