Effect of Emblica officinalis (Amla) on Monosodium Glutamate (MSG) Induced Uterine Fibroids in Wistar Rats

 

Inshah Ahmed¹, Nishat Ahmed², Saleha Ahmed³, Dr. Fazil Ahmad⁴, Dr. Abeer Mohammed Al-Subaie⁵

¹Department of Pharmacology, Nizam Institute of Pharmacy, Deshmukhi (V), Pochampally (M),

Behind Mount Opera, Yadadri Bhuvanagiri (Dist) - 508284, Telangana, India.

²M. Pharm., Pharmacology, Lecturer, Taibah University, Janadah Bin Umayyah Road, Tayba,

Madinah Al-Munawwarah - 42353, Saudi Arabia.

³Department of Pharmaceutics, MRM College of Pharmacy, Hyderabad, 501510, Telangana, India.

⁴Assistant Professor, Department of Anesthesia Technology, College of Applied Medical Sciences in Jubail,

Imam Abdul Rahman Bin Faisal University - Dammam, Jubail 35816, Saudi Arabia.

⁵Assistant Professor of Haematology, Department of Clinical Laboratory Sciences, College of Applied Medical Sciences , Imam Abdul Rahman Bin Faisal University, P.O.Box-1982 - Dammam,  Saudi Arabia.

 

ABSTRACT:

 

 

INTRODUCTION:

The natural products are of great interest in the process of drug discovery. The therapeutic purpose of plant parts like seeds, bark, root, berries, leaves flowers are in the use of phytomedicines. The use of conventional drugs lack appropriate effects and produces severe side-effects on long-term consumption. Due to the serious adverse effects, a lot of research has been started in the field of Ethno pharmacognosy.^(1-3).

There is a growing worldwide interest in the use of phytopharmaceuticals as complementary or alternative medicine, either for the prevention or treatment of many diseases. According to (WHO) World Health Organisation, the world’s population relies more than 80% on traditional medicine for the needs of primary health care^(4-7).. The people found the reason that the thousand of phytochemicals are safe with severe less effect an effective. When the time of ancient like Ayurveda, Siddha, and Unani people use to compare the lower cost when compared with synthetic drugs and due to their less adverse effects. In medicinal properties, the plant has synthesized some metabolites like glycoside, flavonoids, volatile oils, alkaloid, tannins and contain vitamins and minerals^(8-10)..

 

METHODOLOGY:

The fruit of Emblica officinalis (Amla) were collected from local area Hyderabad, Telangana and authenticated in, Department of Botany. The fruit of E. officinalis is taken (300g) and was extracted with using 70% ethanol (3000mL) for 4 h at 80°C in a reflux apparatus. The process was repeated once, and the extracts were combined and filtered through a membrane filter. Once the extraction is done the concentrated solution is taken into beaker and heated on the hot plate to evaporate the excess water. Other method which is very commonly used to make the Amla is taken and add it into hot boiling water and keep it to boil for 20-30 seconds, then sieve it dry it to get the crude extract. The samples were lyophilized to yield a dark yellow powder. The yield of E. officinalis extracts was 18.8%. Synthetic glutamate Monosodium glutamate (MSG) was Obtained from a market for use in the study.

 

Figure 1: Experimental Rats

 

A stock solution was prepared by dissolving MSG granules in 500 Ml Distilled water use. From this and based on the animals weight that morning, the 100 mg/kg dosages were administered to the animals in groups 2 and 3 as part of the 5.0 m L volume used for gastric intubation.

EXPERIMENTAL ANIMALS:

Twenty wistar rats weighing 180-300g were obtained from disease free stocks maintained in the animal House of the Department of Pharmacology at the Nizam Institute of Pharmacy College, Affiliated to JNTU Hyderabad, Telangana.

 

The animals were randomly assigned into four Study groups on the basis of average body weight . Each rat in a study group was individually housed in a stainless cage with plastic bottom grid and a wire screen top. The animal’s room was adequately ventilated and kept at a room temperature and relatively humidity of 29±2^oC and 40-70%, respectively, with a 12 h natural light-dark cycle. Animals were with water and rats chew Good hygiene was maintained by constant Cleaning and removal of feces and spilled from Cages daily. All animals experiments were Approved by the Animal Care and use committee.

 

DRUGS AND CHEMICALS:

Anti fibroid drug (Combined Contraceptive Pills) is obtained from the pharmacy. Monosodium Glutamate is obtained from the market. Methanol, Distilled water, Chloroform and other chemicals for phytochemical screening.

 

ACUTE TOXICITY STUDY:

The acute oral toxicity study was performed according to the OPPTS (Office of Prevention, Pesticide and Toxic Substance (00) the different extract were suspended ZX using. All the extracts of Amla were safe at a dose of 10mg/kg, po., and one tenth of this dose was selected for evaluation of the anti fibroidal activity. Ethical clearance for performing experiments on the animals was obtained from Institutional Animal Ethical Committee (IAEC).

 

EXPERIMENTAL MODELS:

MONOSODIUM INDUCED FIBROIDS IN THE FEMALE RATS:

Table No.1: Drug Dosages

 

All the animals were fed on the basal diet and water ad libitum and they were maintained under healthy conditions of humidity, temperature (20-25 C) and the light (12-h light dark cycle) for one week before starting the experiment to acclimatization. After acclimatization period, the animals were randomly assigned into three study groups Group-I, Group-II, Group-III, Group-IV, Group-V on the basis of average body weight. The extract of the Emblica officinalis were administered po (Per Oral)., The preparation of monosodium Glutamate is also administered as per the group, the drug will be administer by IP (Intra Peritonial) The administration were for 45 days. After that the blood samples from the animals were collected and examined. Twenty four female wistar rats weighing 180-300g were divided into group of four. Group A was the no treatment group (Control). Groups B and C and D were treated with Monosodium glutamate (inducer), Emblica officinalis (test drug) and combined contraceptive pill (Standard drug). Treatment with MSG was conducted for 30 days followed by Amla after which total protein, plasma cholesterol and estradiol were determined. The animals in group is in control receives of 5.0ml distilled water via gastric incubation. And in another groups of 2 and 3 the animals treated with 100mg MSG/Kg with combination of 10mg/kg Amla and in a total volume of 5.0ml vehicle. Where, however the animals were treated with MSG only for 30 day before the commencement of treatment with Emblica officinalis (Amla) extracts and these experiment lasted for 60 days.

 

SAMPLE PREPARATION:

The animals were euthanized by inhalation overdose of chloroform. Blood was collected by cardiac puncture into EDTA sterilized sample bottles. Serum was prepared by centrifugation (6000xg, 30 min) and used for the analysis of serum total serum estradiol (estrogen) serum total protein and serum Lipid Profile.

 

DETERMINATION OF ESTRADIOL (ESTROGEN), SERUM TOTAL CHOLESTEROL, SERUM TOTAL PROTEIN:

Estradiol was determined with modification of an Enzyme Immunoassay (EIA) described by(Meyer et al., 1997) Briefly, 4 Ml of serum samples were adjusted to pH of 3.5 with acetic acid and extracted with 12 m L of diethyl ether (pH 3.5), evaporated and re-extracted with diethyl ether (pH 3.5). The residue was dissolved in 12 m L of assay buffer (40 m M PBS, 0.1% BSA (Bovine serum albumin), pH 7.2) and pooled, resulting in 3.2 m L in PBS (pH7.5) after evaporation, the sample was dissolved in 12 m L of 100% methanol. The content of estradiol in 4ml of each serum was analyzed in triplicate by an enzyme immunoassay described by Meyer et al. (1997). This analyte was identifying by retention time (11.4 min) and the specific antigen-antibody reaction.

 

Serum total cholesterol was determined with the method of Brown and Goldstein (1984). In this assay, cholesterol was extracted from the serum with ethanol. The extract was then reacted with a solution of FeCl₃ dissolved in phosphoric acid and the resulting colour was read in a spectrophotometer 12At 550 nm against a reaction blank.

Serum total protein was determined. Briefly, 0.5 m L of the serum sample solution was pipette into test tubes and 1.0 m L distilled water added to bring the volume to 1.5 Ml in each tube. Tube1 (the blank) received 1.5 m L distilled water. The suspension was mixed and 0.2 m L of 5% sodium deoxycholate (DOC) in 0.01N KOH was added and mixed to make the Suspension more soluble. Then, 1.5 m L of biuret reagent (1.50 g CuSO₄. 5H₂O, 6.0 g sodium potassium tartrate and 300 m L of 10% NaOH perliter) was added (including the blank). The tubes were mixed in a vortex mixer and incubated at 37^oC for 15 min and the absorbance read at 540nm against the blank (tube 1) in a 6400/6405 spectrophotometer. The concentration of the standard Bovine Serum Albumin (BSA) was 2mg/mL.

 

STATISTICAL ANALYSIS:

Were Data is collected and expressed as means ± Standard Error Mean (SEM) and the student ‘t’ test were used for analysis. Values of p<0.01 were regarded assignificant.

 

RESULTS:

Monosodium induced uterine fibroids: The ethanol extract of Emblica officinalis (Amla) showed a significant reduction of in the uterine fibroids when compared with the control group and the standard.

 

GROUP 1: Effect of Amla and standard drug (Combined contraceptive pills) on total serum protein level against MSG induced fibroids

 

Figure No 2: Graphical representation of serum total protein

 

Effect of Amla and standard drug (Combined contraceptive pills) on total serum protein level against MSG induced fibroids. C is the control group. Each column represents Mean ± S.E.M of 6 rats. p< 0.001 compared with the control group (one-way ANOVA followed by Dunnett’s post hoc test).

 

 

 

 

Table no.2: Effect of Amla and standard drug on total protein level

 

GROUP 2: Effect of Amla and standard drug (Combined contraceptive pills) on cholestrol level against MSG induced fibroids

 

Table no. 3: Effect of Amla and standard drug on cholesterol level

 

 

Figure 3: Graphical representation of amla and standard drug on cholesterol level against MSG

 

Effect of Amla and standard drug (Combined contraceptive pills) on cholestrol level against MSG induced fibroids. C is the control group. Each column represents Mean ± S.E.M of 6 rats. p< 0.001 compared with the control group (one-way ANOVA followed by Dunnett’s post hoc test).

 

GROUP 3: Effect of Amla and standard drug (Combined contraceptive pills) on estrogen level against MSG induced fibroids

 

Table No. 4: Effect f Amla and standard drug on estrogen level

 

Figure 4: Graphical representation of Amla and standard drug on estrogen level against MSG induced fibroids

 

Effect of Amla and standard drug (Combined contraceptive pills) on estrogen level against MSG induced fibroids. C is the control group. Each column represents Mean ± S.E.M of 6 rats. p< 0.001 compared with the control group (one-way ANOVA followed by Dunnett’s post hoc test).

 

DESCRIPTION OF RESULTS:

The results demonstrated the effect of Amla, standard drug (combined contraceptive pills) on MSG induced fibroids. Amla and the standard drug cause significant decrease in the serum protein level in rats with probability p < 0.001. In the presents results of treatments on serum total cholesterol. The results showed that treatment with Amla, standard drug (combined contraceptive pills) on MSG induced fibroids. Amla and the standard drug cause significant decrease the cholestrol levels in rats with probability p < 0.001. On these it summarizes the effects of the treatment on the estrogen (estradiol) levels in the rats. The results showed that there was significant increased in the levels of estrogen (estradiol), in the MSG treated hosts relative to those levels in the standard group and in the controls. In the group treated with Amla, estrogen levels were ameliorated with the p < 0.001. The result of this study is shown the possibility of treating women with fibroids and also for extending periods of time without need of surgery.

 

DISCUSSION:

The present study demonstrated the effect of Amla on uterine fibroids. The severity of symptoms, size and location of lesion, patients age, chronological proximity to menopause has to be considered for the therapy individualization. The treatment options range from conservative measures (reassurance observation) to radical procedures (hysterectomy). There is no definitive therapeutic agent developed for long term treatment of fibroids but some drugs like GnRH analogues (leuprolide and goserelin), progesterone-receptor modulators such as ulipristal had effectively reduced uterine leimyoma volume and also the symptoms like heavy menstrual bleeding and loss of haemoglobin was restored.

 

CONCLUSION:

In conclusion, the results demonstrated that MSG has increased the levels of total protein, cholesterol and estrogen resulting in the formation of fibroid. However, treatments with Amla extracts near-completely mitigated the levels of total protein, cholestrol and estrogen induced by the MSG alone. This effect could be attributed to the phenolic and other constituents and one of the mechanism could be balancing of oxidative stress by acting as anti-oxidant. Further studies are needed to confirm the activity and its mechanism.

 

REFERENCES:

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9.      Brower et al., 2000

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Accepted on 28.08.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(6):2535-2539.