PROTECTIVE EFFECT OF OKRA, ABELMOSCHUS MOSCHATUS SEED EXTRACT ON DEVELOPING BRAIN OF RATS DURING PRE- AND POST-NATAL FLUORIDE EXPOSURE

PROTECTIVE EFFECT OF OKRA, ABELMOSCHUS MOSCHATUS SEED EXTRACT ON DEVELOPING BRAIN OF RATS DURING PRE- AND POST-NATAL FLUORIDE EXPOSURE

Kurmeti Sudhakar, Mesram Nageshwar and K. Pratap Reddy^*

Department of Zoology, Osmania University, Hyderabad - 500007, Telangana, India.

ABSTRACT: This study reports protective effect of Abelmoschus moschatus seed extract against sodium fluoride (NaF) induced alterations in behavior correlated with neurochemical changes in developing brain of rats. Excessive intake of fluoride during pregnancy cross the blood brain barrier (BBB) and cause adverse effects on neonatal development. As the BBB in fetuses, neonates and infants is immature, it cannot provide protection against the entries. The pregnant wistar rats were randomly categorized into six groups of five animals each. Group I is of control rats received normal tap water. Group II is NaF exposed group with 20 ppm (or 20 mgkg^-1 body wt.) in their drinking water. Group III rats were treated with A. moschatus aqueous extract (AMAE) (300 mgkg^-1 body wt./day/rat) along with NaF water (20 ppm). Group IV rats were treated with A. moschatus ethanolic extract (AMEE) (300 mgkg^-1 body wt./day/rat) along with NaF water (20 ppm).Group V and VI rats were treated with AMAE (300 mgkg^-1 body wt./day/rat), and AMEE (300 mgkg^-1 body wt./day/rat) respectively. On 1^st, 7^th, 14^th, 21^st and 30^th day (postpartum days), the pups were sacrificed to assess oxidative stress markers (LPO, CAT), and measured body weight, brain weight, BSI, and estimated protein content of brain tissue of all experimental groups. On post-natal day 21 and day 30 pups behavioral activity (rota rod, hot plate test) was measured. Fluoride exposure significantly increased lipid peroxidation and decreased the activity of catalase, and decreased body and brain weight, BSI and also protein content of the brain of pups indicating oxidative stress and inhibited antioxidant system and protein synthesis which were reverted on administration of AMAE and AMEE against NaF intoxication. The altered behavioral responses on NaF exposure were also reversed to that of control. Hence, this study proves the vulnerability of developing brain to fluoride toxicity during development and growth and protection offered by AMAE and AMEE towards neurotoxicity of NaF.

Sodium fluoride, Oxidative stress, Neurotoxicant, Abelmoschus, Behavior

INTRODUCTION: Prolonged ingestion of fluoride through various sources, mainly drinking water caused fluorosis ¹. Fluorosis, as a global public health problem, has been receiving wide and deserving attention in recent years. Prolonged consumption of fluoride in excess during gestation cause adverse effects on neonatal development ². The mechanisms of maternal fetal transmission of fluoride are poorly understood.

Therefore, the present study focused on maternal exposure of fluoride and it’s effects on developing CNS and it’s amelioration with extract of okra seeds. Fluoride is known to cross the placenta ^{3, 4} and the blood brain barrier ⁵ and ultimately increase fluoride levels in fetal brain tissue. The blood brain barrier in the embryo, fetus, and newborn is “immature” that means it is poorly developed, leaky, or even absent, rendering the developing brain more vulnerable to fluoride entering the fetal circulation from the mother ⁶. Once it enter into the brain it can cause adverse effects on the brain cell ultra structure, metabolism, enzymes, the oxidant-antioxidant status and on neurotransmitters and thus overall metabolism of brain ^{7, 8}. The maternal exposure to fluoride and fluoride insobriety at the early periods of life is shown to cause more prominent effects on the oxidant-antioxidant status in the brain than the fluoride exposure at the later stages of life ^{9, 10}. Fluoride increases production of reactive oxygen species (ROS), lipid peroxidation, and decreased catalase activity which have been considered in pathogenesis and recent studies suggest that oxidative stress is a possible pathologic mediating factor in fluoride toxicity ^{11, 12}.

Exposure to fluoride during early developmental stages of life results in long-term irreversible consequences with their structure and function and account for qualitative differences in age related susceptibility ¹³. The neuronal growth-spurting in rats occurs in the first 3 weeks of postpartum ¹⁴. During this period the brain undergoes many profound structural and functional transformations, which makes vulnerable to fluoride ¹⁵.

The high intake of plant products is associated with a reduced risk of a number of chronic diseases ¹⁶. Earlier reports on natural products which aid in protection against NaF induced behavioral alterations are: Curcumin ¹⁷, Quercetin ¹⁸, Rutin (extracted compound of Okra) ¹⁹, Vitamin A ²⁰, and Spirulina platensis ²¹. These natural products reverse behavioral alterations which are induced on NaF exposure. These beneficial effects have been moderately attributed to the compounds which possess antioxidant activity.

Abelmoschus moschatus L., (Okra) is one of the most commonly known and utilized species of the family Malvaceae. The major antioxidants of vegetables are vitamins C and E, carotenoids, and phenolic compounds, especially flavonoids ²². Nutritionally, the richest part of the okra plant is the dried seed. Okra seed is rich in high quality protein especially with regard to its essential amino acids ²² and unsaturated fatty acids such as linoleic acid ²². Seeds are rich in phenolic compounds with derivatives, catechin oligomers and hydroxyl-cinnamic derivatives. The nutrients content of Okra seed showed that 21% protein, 14% lipids and 5% ash. Proteins play a particularly important role in human nutrition ²². Okra is a popular health food due to its high fiber, Vitamin C, and folate content and also a good source of calcium and potassium. High fiber, “helps to stabilize blood sugar by regulating the rate at which sugar is absorbed from the intestinal tract” ²³. Okra seeds will make available the essential energy to the body and important antioxidants that could boost immune system and prevent diseases ²⁴. The neuroprotectant effects of extract of Okra against fluoride induced behavior and neurodegenerative changes in brain of adult rat were reported in our earlier studies ²⁵. In view of this, the efficacy of AMAE and AMEE in reducing fluoride induced neurobehavioral, and neurochemical alterations in rats of pre and post-natal fluoride exposed is reported in this paper.

MATERIAL AND METHODS: Healthy albino wistar rats (Rattus norvegicus) weighted between 180 to 200 gm were assigned randomly to experimental groups and housed 2 females and 1 male for cage (polypropylene cages) to allow breed. Light cycles were maintained as 12 Light: 12 Dark hours (6.00 AM to 6.00 PM), and temperature between 22 ± 2^oC. The rats were maintained on standard diet and water was supplied ad libitum. After their pregnancy was confirmed, from the first day pregnancy, the females made into six groups and allowed to drink on fluoridated water at the rate of 20 ppm NaF and also Abelmoschus moshatus seed extract was given to the rats at the rate of 300 mgkg^-1 body weight as follows;

Experimental Groups:

• Group I: Received normal tap water
• Group II: Fed on fluoridated drinking water (20ppm)
• Group III: NaF (20 ppm) + Abelmoschus moschatus seed aqueous extract (AMAE) (300mgkg^-1 wt.)
• Group IV: NaF (20 ppm) + Abelmoschus moschatus seed ethanolic extract (AMEE) (300mgkg^-1 wt.)
• Group V: Abelmoschus moschatus seed aqueous extract (AMAE) (300 mgkg^-1 wt.) only.
• Group VI: Abelmoschus moschatus seed ethanolic extract (AMEE) (300 mgkg^-1 wt.) only.

Chemicals: Potassium dichromate, glacial acetic acid, Thiobarbituric acid (TBA), H₂O₂, sodium carbonate, sodium potassium tartrate, Folin reagent etc. were purchased from local laboratory chemical suppliers.

Physical Parameters:

Body Weight: The rat pups were weighted at the age of post-natal Day 1, Day 7, Day 14, Day 21 and Day 30 and noted down the readings.

Brain Weight: The same pups were killed by decapitation and their brains were dissected out. These brains weighted and noted down the values.

Brain Somatic Index (BSI): For calculating the brain - somatic index, the weight of the individual rat was noted and the brain was removed carefully and weighed on an electronic weigh balance after removing the blood with tissue paper. BSI was calculated by using the value of brain weight of control rat divided by the same rat body weight value and multiplied with 100 (brain weight / body weight × 100). This formula was applied to all experimental groups to calculate BSI. The Brain - Somatic Index of the rat pups were calculated by the use of equation cited by Ashwini ²⁶.

Brain Somatic Index = Brain weight × 100 / Body weight

Behavioral Assessments:

Rota Rod: The rota rod test, in which rat pups must balance on a rotating rod, is widely used to assess motor deficit in neurodegenerative disease models (in rodents). Performance is measured by the duration that an animal stays on the rod as a function of rotating rod speed. In the present study, the instrument (Dolphin^TM instruments) had incremental fixed-speed (about 30 rpm) rotating rod. Before experimental testing, rat pups were trained to run on the rotating rod in 3 training trials per day for three consecutive days with a constant speed of 30 rpm. For this purpose, animals were kept on the rotating rod for 2 min, and time of their first falling off and the frequency of falling off the rod are recorded for each rat. After the training period, on the day of testing, the performance of the rats was measured as maximal time spent on the rod at 30 rpm before falling off and endurance time noted in min ²⁷.

Hot Plate Test: The hot plate test is used for evaluating thermal pain sensitivity. The hot plate test evaluates thermal pain reflexes due to footpad contact with a heated surface. An apparatus consisting of an aluminium plate that is heated and cooled by Peltier elements in contact with its lower surface. During the experiment, the rat was introduced into Remi hot plate kept at 52 ± 0.5 °C. Hot-plate latencies are determined as described by placing each rat pup on a hot plate and observing the occurrence of a nociceptive response (licking of a hind paw, jumping). The time of exhibiting these behaviors was noted as response time and recorded in seconds. To observe the rat behavior, observation area covered with a colourless plastic cylinder was placed on the hot-plate. In order to avoid possible tissue injury, a cut-off time of 12 seconds was used to avoid skin damage. One hot plate test was carried out for each rat ²⁸.

Oxidative Stress Markers: Lipid per-oxidation and catalase were assessed.

Lipid per-oxidation (LPO): The LPO was assessed by the method of Bhuyan ²⁹. This assay is based on the reactivity of an end product of lipid peroxidation, malondialdehyde (MDA) with TBA to produce a pink adduct, which is measured at 533 nm on spectrophotometer. The Levels of lipid peroxidation products expressed as µmol of MDA/gr. weight of tissue.

Catalase: Catalase activity was estimated by measuring the rate of decomposition of H₂O₂. Disappearance of peroxide in the presence of enzyme source is the basis for assay which is followed spectrophotometrically at 505 nm. One unit decomposes one micromole of H₂O₂ per minute ³⁰.

Protein Estimation of Brain: Brain tissue total protein content was estimated by the method of Lowry ³¹. The principle of Lowry method is the peptide nitrogen(s) with the copper [II] ions under alkaline conditions and the subsequent reduction of the Folin - Ciocalteau phosphomolybdic phosphotungstic acid to heteropolymolybdenum blue by the copper catalyzed oxidation of aromatic acids. The color was read at 540 nm. Protein content in the brain tissue expressed as mg of protein / gm weight of tissue.

Per cent of change between groups was calculated by the formula of;

% change =    Experimental – Control × 100 / Control

Data Analysis: Statistical significance was determined by one way analysis of variance. Differences between means were determined by t-test.

RESULTS:

Body Weight: The body weight was progressively decreased (Table 1) in NaF treated rats from post-natal day 1 to post-natal day 30 rats as compared to control. Among these experimental days the maximum per cent (-14.34%) of decreased body weight was recorded in post-natal day 30 NaF treated one.

Furthermore, the reverted body weight was noticed in F+AMAE and F+AMEE compared to fluoride alone treated group of respective age and it was similar to control group.

TABLE 1: EFFECT OF ABELMOSCHUS MOSCHATUS SEED EXTRACT ON BODY WEIGHT, BRAIN WEIGHT, BRAIN – SOMATIC INDEX (BSI) OF RAT PUPS (FROM POST-NATAL DAY 1 TO POST - NATAL DAY 30) EXPOSED TO NaF