Description:FORM 2

THE PATENTS ACT,
(39 OF 1970)
THE PATENT RULES, 2003.

COMPLETE SPECIFICATION
(SECTION 10 AND RULE 13)

Saponin Rich n-Butanolic Fraction of Ziziphus nummularia Leaf Extract against Obesity Induced Alzheimer’s Disease and its neuroprotective effect

The following specification particularly describes the invention and the manner it is to be performed.

Saponin Rich n-Butanolic Fraction of Ziziphus nummularia Leaf Extract against Obesity Induced Alzheimer’s Disease and its neuroprotective effect

FIELD OF THE INVENTION
The present invention relates to compositions of Saponin Rich n-Butanolic Fraction of Ziziphus nummularia Leaf Extract against Obesity Induced Alzheimer’s Disease and its neuroprotective effect

BACKGROUND OF THE INVENTION
Alzheimer’s disease (AD) is an irreversible, progressive neurodegenerative brain disease that slowly destroys memory and thinking skills and eventually even the ability to carry out the simplest tasks. It is associated with localized loss of cholinergic neurons, mainly in the hippocampus and frontal cortex of the brain disclosed in Amartya D, Bala N, Pallab DG. Alzheimer’s disease and its Management. Int. J. Res. Pharm. Biomed Sci. 2011, 2:1439-1443, and is the most common cause of dementia among people of age 65 and older and disclosed in Rang HP, Dale MM, Ritter JM and Flower RJ. Rang and Dale’s Pharmacology, 2007, 6th edition: 514-517.
The prevalence of AD increases exponentially with age, affecting approximately 7% of individuals ages 65 to 74 years, 53% of those ages 75 to 84, and 40% of persons ages 85 years and older. Onset can be as early as age 40 years, resulting in the arbitrary age classifications of early onset (ages 40 to 64 years) and late-onset (ages 65 years and older), AD is believed to develop in response to a combination of genetic and nongenetic factors, which may be different in different individuals disclosed in Claudine F, Mary TH, Rita R. Advances in Diagnosis and Treatment of Alzheimer’s Disease. Hosp Physician. 2002: 47 – 54.
The pathophysiological factors responsible for AD include β-Amyloid plaque and neurofibrillary tangle formation, decreased Acetylcholine levels, Glutamate toxicity in neurons, oxidative stress, chronic inflammation and Genetic predisposition. Other risk factors that contribute to AD are Age, Obesity, Unhealthy Eating Habits, Diabetes, Hypertension, High Cholesterol, Insulin Resistance, History of Head Injury is disclosed in Jana P, Ambroza P, Michal B, Veronika P, Omar S, Hana T, et al. Epidemiological of and risk factors for Alzheimer‘s disease: A review, Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2012; 156: 108–114 .
Recently AD was added to the obesity-related diseases. Obesity is a chronic metabolic disorder caused by an imbalance between energy intake and expenditure is disclosed in Changhyun R, Uhee J. Screening of Crude Plant Extracts with Anti-Obesity Activity. Int. J. Mol. Sci. 2012; 13: 1710-1719.
Over the last decade, several magnetic resonance imaging (MRI) and computed tomography (CT) studies demonstrated a higher BMI and/or waist-to-hip ratio in middle-aged individuals associated with a reduction in brain volume in the frontal temporal lobes, anterior cingulate gyrus, and hippocampus. This study reported an inverse association between various obesity indicators (BMI, waist circumference, waist-to-hip ratio, and abdominal fat and brain volume is disclosed in Rana A, Avinash P, Christian LH. Influence of Obesity on Neurodegenerative Diseases. In Intech. Neurodegenerative Diseases. 2013; p. 382-402.
Obesity results in the AD onset and progression by altering the normal physiological processes in the body and their by promoting various pathophysiological pathways that effect the brain function which include- The more the fat person carrying around, the more resistant body becomes to insulin, to try to remedy this insulin resistance and to overcome hyperglycemia body makes more insulin, if the cycle continues the end result is Hyperinsulinemia. In the CNS, Insulin acts like a neuroprotective and neurotrophic factor, since it promotes neuronal survival and also plays a key role in cognitive function by modulating CNS concentration of neurotransmitter associated with important roles in cognition such as acetylcholine is disclosed in Sung MS, Hong JS, Inhee MJ. Insulin Resistance and Alzheimer’s Disease. In Intech: Mark Z, editor. Topics in the Prevention, Treatment and Complications of Type 2 Diabetes. 2011; p. 53-74.
The elevated insulin levels and central insulin resistance are implicated in the brain cell’s failure to clear beta-amyloid, increased Aβ toxicity, Tau hyperphosphorylation, oxidative stress, neuroinflammation, DNA damage, mitochondrial dysfunction, which finally leads to neurodegeneration and AD is disclosed in Karen FN, Leonel R, Leonardo P.N, Gonzalo F, Paula R, Ricardo BM. Insulin Resistance and Alzheimer´s Disease: Molecular Links & Clinical Implications. Curr Alzheimer Res. 2008; 5: 1-10.
Adipose tissue is the site where the excess fat gets accumulated. However, it is now recognized as an endocrine tissue producing a number of inflammatory-related factors, acting at a physiological level. As obesity takes place, the release of inflammatory cytokines by activated macrophages in visceral adipose tissue becomes higher. Several recent studies prospectively assessed the predictive value of elevated pro-inflammatory cytokines for the risk of developing AD in cognitively intact individuals or for aggravating AD symptoms in patients who were already diagnosed with the disease is disclosed in Rita B, Flora L, Serafino R, Nicoletta C, Andrea F. Alzheimer’s Disease Promotion by Obesity: Induced Mechanisms—Molecular Links and Perspectives. Curr Gerontol Geriatr Res. 2012; 2012: 1-13.
Hyperlipidemia/hypercholesterolemia is the major vascular risk factor for narrowing of blood vessels. The occlusion of blood vessels that supply blood to brain results in cerebral ischemia, due to which the neurons become deprived of and ultimately get destroyed. After cerebral ischemia, necrotic neuron death in the local ischemic area, microglia of the brain get activated, their morphology is changed, and they start to secrete proinflammatory cytokines such as interleukin (IL)-1, tumor necrosis factor α (TNF- α), and interferon γ (IFN- γ ) and starts the inflammatory cascades disclosed in Ashu A, Parveen A, Mamta K, Sunil K. Cerebral Ischemic Stroke: Sequels of Cascade. Int J Pharm Bio Sci. 2010; 1: 1-24.
The increased cholesterol levels/hypercholesterolemia in obese individuals leads to increased cleavage of APP and increased production and deposition of Aβ, the hall mark of Alzheimer’s disease is disclosed in Luigi P, Rudolph E, Tanzi, Dora MK. Alzheimer’s disease cholesterol connection. Nat Neurosci. 2003; 6: 345-351.
For the present study, we have selected a plant called Ziziphus nummularia belonging to family Rhamnacae. The leaves of the plant are traditionally used to reduce obesity and the leaf extract was reported to have hypolipidemic, hypoglycemic and anti-inflammatory properties. Saponins are the main constituents present in the leaf, they cause a depletion of body cholesterol levels by preventing its reabsorption into the system and by increasing its excretion disclosed in Omale J, Haruna HU. Hypocholesterolemic Effects of Nauclea latifolia (Smith) Fruit Studied in Albino Rats. Amer J Trop Med Pub Health. 2011; 1: 11-21.
Saponins were also found to inhibit the sweet taste receptors in animals and humans resulting in reduced sugar intake and thereby reducing obesity in diabetic or overweight is disclosed in Suttisri R, Lee IS, Kinghorn AD. Plant-derived triterpenoid sweetness inhibitors. J Ethnopharmacol. 1995; 47: 9-26, saponin rich n-butanolic fraction was isolated from ethanolic leaf extract of Ziziphus nummularia and its effect on high fat diet fed obese mice to reverse the obesity associated pathophysiologic processes underlying AD onset and progression was investigated.
Considering the above facts and the need in the public interest for an ideal herbal preparation of formulation that can used for the treatment of obesity and Alzheimer’s disease. An attempt was made by the inventors of the present application to prepare the composition of herbal preparation and using the typical process which can scaled up in larger quantities if required. Additionally, the invention disclosed in the present application was surprisingly found to be superior and has a potential treatment of obesity and Alzheimer’s disease.

SUMMARY OF THE INVENTION
An embodiment of the present invention relates to a preparation of the composition of herbal extract and manufacturing methods thereof.
An embodiment of the present invention relates to a preparation of the composition of the herbal extract of n-Butanolic Fraction of Ziziphus nummularia Leaf and being used for the treatment of obesity and Alzheimer disease via oral route of administration.
An embodiment of the present invention relates to a formulation for preparation of herbal n-butanolic leaf extract.

DETAILED DESCRIPTION:
An embodiment of the present invention relates to a preparation of the composition of herbal extract and manufacturing methods thereof.
An embodiment of the present invention relates to a preparation of the composition of the herbal extract of n-Butanolic Fraction of Ziziphus nummularia Leaf and being used for the treatment of obesity and Alzheimer disease via oral route of administration.
An embodiment of the present invention relates to a formulation for preparation of herbal n-butanolic leaf extract.

MATERIALS AND METHODS
The leaves of Ziziphus nummularia were collected in the month of July, 2013 from the shrubs located in open fields around Kakatiya University, Warangal, Telangana, India. Sample was authenticated by a botanist Prof. V S Raju, Department of Botany, Kakatiya University.
1. Preparation of extract. The fresh leaves of Ziziphus nummularia were dried under shade, sliced into small pieces and ground into powder with mechanical grinder. The powder was passed through sieve no.30 and stored in a container. The dried leaf powder of Ziziphus nummularia was defatted with petroleum ether in a Soxhlet apparatus by hot percolation. The defatted powder material (marc) thus obtained was further extracted with Ethanol (95% v/v) by maceration. The extract was concentrated into a semi-solid material using rotary evaporator at 50°C as per the method described in Rajasekaran S, Velmurugan C, Vetriselvan S, Rusliza B, Muthuramu T. Phytochemical And Pharmacological Evaluation Of Leaves of Zizyphus Nummularia (Burm.F.)Wight&arn. Int. J. Phytopharmacology. 2012; 3: 299-303.

Separation of saponin rich n-butanolic fraction: The remaining extract was partitioned with n-butanol, 1:1 (v: v) overnight at room temperature using a separator funnel. The upper n-butanol extract was collected in a glass flask and the lower aqueous extract was collected and further partitioned with n-butanol two more times to increase the yield of crude saponin extract. The butanol extracts were pooled and evaporated to remove the butanol under vacuum at temperature below 50°C using the rotory evaporator to obtain solid dry mass of saponin rich n-butanolic fraction of ethanolic extract of leaves of Ziziphus nummularia (SBZN).

Experimental animals
Swiss Albino Mice (25-30 gms) were used for the study. They were maintained at 20±2°C and relative humidity of 45 to 55% water and under standard environmental conditions (12 hours light 12 hours dark cycle). They had free access to standard food pellets and water ad libitum. The experiments were conducted after obtaining approval from institutional animal ethical committee (1047/ac/07/CPCSEA, Dated 24-04-2007) and care of animals was taken as per guidelines of CPCSEA, Department of Animal Welfare, and Government of India. Animals were quarantined and acclimatized to laboratory conditions for 4 days prior to study initiation.

Experimental design
Phytochemical tests for saponins
Foam test - The extract was diluted with 20ml of distilled water and was shaken in a gradual cylinder for 15 min. A 1cm layer of foam indicates the presence of saponins.
Haemolysis test - One drop of extract and one drop of blood were placed on the glass slide. Haemolytic zone appeared indicates the presence of saponins as per method disclosed in Kokate CK. In: Practical Pharmacognosy, Preliminary Phytochemical Screening, 1986, 1st edition: 111.
Acute Toxicity Test- Acute toxicity of SBZN was carried out as per the OECD guideline 425. A limit test was performed using healthy albino mice of either sex (25-30g) maintained under standard dietary conditions. Prior to dosing, animals were fasted for 3-4 hours and the dose for each animal was determined based on body weight. Initially SBZN was administered to one animal in a single dose of 2000 mg/kg by using an oral gavage. After the administration, food was withheld for a further 1-2 hours, then the animals were observed for first 24hrs and further 14 days for any signs of behavioral changes, toxicity, changes in body weight and mortality as disclosed in The Organization for Economic Co-operation Development (OECD). The OECD guideline for Testing of Chemicals 425, adopted 3rd October 2008.
Inducing Obesity in mice
Animals to be grouped under Pathological control, test-1 and test-2 were provided with high fat diet for 6 weeks to induce obesity, whereas the normal control group with normal pellet chow and water ad libitum.
Composition of High fat diet (100gms of diet consists of)
Vanaspathi-10gms + Ghee-10gms + All-purpose flour-30gms + Milk Powder-10gms + Groundnut Powder-20gms + Sucrose-10gms + Coconut Powder-10gms. The diet (contains substantial amount of sugar and fat) was formulated as per the unhealthy eating habits of people and was meant to simulate the human western diet. (Figure No: 1)
After inducing obesity the animals (n = 24) were divided into four different groups of 6 animals per each group with uniform weight distribution
Group 1 Normal control Distilled Water
Group 2 Disease control High Fat Diet
Group 3 Test-1 SBZN(100mg/kg) oral
Group 4 Test-2 SBZN(300mg/kg) oral

Behavioral Tests
All the animals were trained for 2 days before drugs administration.
Rectangular Maze Test
The maze consists of completely closed rectangular box with an entry and reward chamber partitioned with wooden slats into blind passages leaving just twisting corridor leading from the entry to the reward chamber. On the first day all the mice were familiarized with rectangular maze for a period of 10 min. This was known as training session. On the 3rd day the mouse was placed in the entry chamber and the timer was activated as soon as the mouse leaves the entry chamber. The time taken for the mouse to reach the reward chamber was taken as the latency time. 4 readings were taken and an average of reading gives learning score. Lower scores indicate efficient learning and higher scores indicates poor learning in animals.
Morris Water Maze Test
Morris water maze was used to assess learning and memory in experimental mice. Method was carried out in a circular pool (90 cm in diameter and 50 cm in height) of water with a featureless inner surface as per method disclosed in Morris R. Developments of a water-maze procedure for studying spatial learning in the rat. J. Neurosci. Methods. 1984, 11: 47–60.
The 1st day of the experiment was dedicated to swimming training for 60 sec in the absence of the platform. During the 4 consecutive days the mice were given the trial session with the platform in place. Once the mouse located the platform, it was permitted to remain on it for 10 sec. If the mouse did not locate the platform within 120 sec, it was placed on the platform for 10 sec and then removed from the pool. One day after the final training trial sessions (on day 5), mice were individually subjected to a probe trial session in which the platform was removed from the pool, and mice were allowed to swim for 120 sec to search for it and the latency time was determined by Manish KS, Sudesh P, Krishan K, Akshay A. Bacopa monniera Attenuates Scopolamine-Induced Impairment of Spatial Memory in Mice. Evid. Base. Compl. Alternative Med. 2011; 2011: 1-10.

Locomotor Activity
Locomotor activity is influenced by most of the CNS drugs in both man and animals. The locomotor activity of drug can be studied using Actophotometer which operates on photoelectric cells which are connected in circuit with a counter when the beam of light falling on photocell is cut off by the animal, then a count was recorded. Animals were placed individually in the activity cage for 10min and the activity was monitored. The test was done 30 min after the drug administration. The photocell count is noted and decrease or increase in locomotor activity was determined.
Pole Climbing Test
When an electrical stimulus is given to animal, it tries to escape from it and move to the near safe place. This equipment is designed in such a way to climb the pole when stimulus is generated. Prior to the experiment, animals were trained. Training and testing were conducted in a 25x25x40 cm chamber that was enclosed in a dimly lit attenuated box. Scrambled shock was delivered to grid floor of the chamber. A 2.8 KHZ speaker and a 28v light were situated on the top of the chamber. A wooden pole 2.5 cm in diameter was suspended by a counterbalance weight through a hole in the upper layer centre of the chamber. The response was recorded when a mice jumps on the pole and activates micro switch. The activation of light and speaker together were used as conditioned stimulus.

Dissection and Homogenization.
On day 15th, after behavioral assessment, animals were scarified by cervical dislocation. The brains were removed and washed with ice cold normal saline. A (10% w/v) homogenate was prepared for each brain separately in 0.1M phosphate buffer (pH 7.4). The homogenate was centrifuged at 3000rpm for 15 minutes and supernatant were separated and used for biochemical estimation as described in Vogel G, Wolfgang HV, Bernward AS, Jurgen S, Gunter M, Wolfgang FV. Drug Discovery and Evaluation. Pharmacological Assays. 2002, 2nd edition: 595-627.

Biochemical Tests
Acetylcholine Esterase (AChE) Estimation
Brain tissue homogenate was incubated for 5 min with 2.7ml of phosphate buffer and 0.1ml DTNB reagent was added. Then the supernatant obtained was added with 0.1ml acetylthiocholine iodide substrate. The resulting yellow color was due to reduction of DTNB by certain substances in brain homogenate and due to non-enzymatic hydrolysis of substrate. DTNB reagent containing solution was used for zeroing the colorimeter. The absorbance was read at 420nm. Cholinergic concentration was spectrophotometrically determined as per method described in Abhinav K, Jogender M, Madhusudana M, Naidu VGM, Gupta YK. Anti-amnesic activity of Vitex negundo in scopolamine induced amnesia in rats. Pharmacology & Pharmacy.2010; 1: 1–8.

Catalase Activity
Hydrogen peroxide (H2O2) solution (2mM/L) was prepared with standard phosphate buffer (pH 7.4). The brain tissue homogenate supernatant was added to 0.6ml of H2O2 solution. Absorbance was determined at 230nm after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide. The percentage scavenging activity was determined as per method described in Kaur IP, Geetha T. Screening methods for antioxidants—a review. Mini-Rev Med Chem. 2006; 6: 305–312.

Thiobarbituric Acid Reactive Substances (TBARS) Assay
The reaction of thiobarbituric acid (TBA) with malondialdehyde (MDA), a secondary product of lipid peroxidation has been widely adopted as a sensitive assay method for measurement of lipid peroxidation in biological fluids. The tissue homogenate (0.5ml) was supplemented with 0.5ml of phosphate buffer and then with 1ml of 10% trichloroaceticacid. The mixture was centrifuged at 3000rpm at 40c for 10 min. the supernatants of the tissue homogenates were incubated with 1 ml of 0.8% w/v of the thiobarbituric acid at 1000c for 15 min. After a cooling period, TBARS concentration was spectrophotometrically determined at 532nm. The levels of lipid peroxides were expressed as nano moles of TBARS. Standard graph was plotted using TEP (1, 1, 3, 3-tetra ethoxy propane) as per method described in Kaur IP, Geetha T. Screening methods for antioxidants—a review. Mini-Rev Med Chem. 2006; 6: 305–312.
DPPH (2, 2-Diphenyl-1-picrylhydrazyl) Assay
The free radical scavenging activity of the test drug was measured in vitro by 1,1-diphenyl-2-picrylhydrazyl(DPPH) assay. About 0.3mM solution of DPPH was dissolved in 100ml ethanol and 1ml of this solution was added to 3ml of the brain tissue homogenate was dissolved in ethanol. The mixture was shaken and allowed to keep at room temperature for 30 min and the absorbance was measured at 517nm using a spectrophotometer. The percentage of scavenging activity was determined.
The % radical scavenging activity was calculated as follows:
% radical scavenging = (Ablank − Asample/Ablank) × 100
Ablank = Absorbance in absence of test sample
Asample = Absorbance in presence of test sample

Estimation of blood glucose by GOD/POD method
Three test tubes were taken and labeled as blank, test, standard. To the blank, 1ml glucose reagent and 0.01 ml distilled water were added. To the test, 1ml glucose reagent and 0.01 ml sample were added. To the standard 1 ml glucose reagent and 0.01 ml glucose standard were added, mixed well and incubated at 37°C for 10 min and then the absorbance was measured at 505 nm as per method described in Trinder. P. Glucose assay: A colorimetric enzyme-kinetic method assay. Annals of Clin Biochem. 1969; 6: 24.

Estimation of total cholesterol by CHOD/PAP method
Three test tubes were taken and labeled as blank, test and standard. To the blank, 1ml of enzyme reagent and 0.01 ml of deionized water were added. To the test 1ml enzyme reagent and 0.01ml of sample were added. To the standard 1ml of enzyme reagent and 0.01 ml of cholesterol standard were added. Mixed well and incubated at 37°C for 5 min. The optical density (O.D) was read at 500 nm against blank as per method described in Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem. 1974; 20: 470-475

Estimation of triglycerides by GPO/PAP method
Three test tubes were taken and labeled as blank, test and standard. To the blank, 1ml of enzyme reagent and 0.01 ml of deionized water were added. To the test 1ml enzyme reagent and 0.01ml of sample were added. To the standard 1ml of enzyme reagent and 0.01 ml of cholesterol standard were added. Mixed well and incubated at 37°C for 10 min. The absorbance of standard and test was read on photo colorimeter at 546 nm against blank as per the method described in Bucolo G, David H. Quantitative determination of serum triglycerides by the use of enzymes. Clin Chem. 1973; 19(5): 476-482.

HISTOPATHOLOGICAL STUDIES
After 14 days treatment, the brains of different groups were perfusion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed and post fixed in the same fixative overnight at 48°C. The brains were then routinely embedded in paraffin and stained with Hematoxylin-Eosin. The hippocampal region was assessed microscopically at 40X magnification. The statistical analysis of data was done by the one way analysis of variance (ANOVA) followed by the Dunnett’s test using Graph Pad Prism 5.0, USA. The probability level less than 0.05 (P<0.05) were considered as significant. Results were expressed as Mean ± SD.
Phytochemical Tests for Saponins
Charecteristic test Observation Inference
Foam test +ve Presence of saponins
Haemolysis test +ve Presence of saponins

Acute Toxicity Test (OECD guideline no. 425)
The LD50 value of the saponin n-butanol fraction of Z. nummularia (SBZN) was found to be greater than 2000mg/kg of body weight. Animals treated with SBZN were free from signs toxicity and no mortality was found up to 2000 mg/kg. Hence two doses 100 and 300mg/kg were selected for present study.
Behavioral Tests
Rectangular Maze Test
The activity of SBZN (100mg/kg & 300mg/kg) was evaluated using rectangular maze. The mice in treatment groups have shown significant (P < 0.05) decrease in transfer latency time on 7th and 14th day when compared to pathological control (obese) group (Figure No. 2). This indicates the learning and memory enhancing activity of a test drug.
Morris Water Maze Test
The activity of SBZN was evaluated using Morris water maze. The mice in treatment groups have shown significant (P<0.01) decrease in transfer latency time compared to pathological control group (Figure No. 3). This indicates the learning and memory enhancing activity of a test drug.

Locomotor Activity
The activity of SBZN was evaluated using Actophotometer. The number of crossings by mice in treatment groups were significantly (P<0.05) increased on day7 and 14 when compared to pathological control group (Figure No. 4). It indicates an increased motor activity in drug treated mice compared to obese mice group.
Pole Climbing Test
The values obtained from the test have shown significant (P<0.05) differences between the SBZN treated groups and pathological control group, the obese mice took more time to reach the pole (latency time) when compared to treated mice (Figure No. 5).

Biochemical Tests
AChE Estimation.
SBZN treatment (100mg/kg & 300mg/kg) significantly (P<0.001) decreased the brain AChE enzyme activity compared to pathological control group (Figure No. 6).
TBARS Assay.
SBZN treatment (100mg/kg & 300mg/kg) significantly (P<0.05) decreased the brain MDA level compared to pathological control group (Figure No. 7). It indicates an increased lipid peroxidation in obese mice brain.
Catalase Activity.
The % H2O2 scavenging activity was decreased in obese mice compared to the normal control group. Significant (P < 0.05) difference has been found between the values of SBZN drug treated groups and pathological control group (Figure No. 8). It indicates an increase in Catalase levels by SBZN.
DPPH Assay.
The % radical scavenging activity was decreased in obese group compared to normal control group. Test drug treated groups have shown significant (P <0.05) difference in the values compared to the disease control group (Figure No. 9). It indicates an increased Antioxidant levels in SBZN treated mice.
Estimation of Blood glucose levels
The blood glucose levels of pathological control group were high compared to test drug treated groups. The SBZN treatment significantly (p<0.05) decreased the blood glucose levels in mice, (Figure No. 10) this indicates the hypoglycemic effect of saponins

Estimation of triglyceride levels
The serum triglyceride levels were found to be high in pathological control group when compared to other groups. The SBZN treatment significantly (P<0.001) reduced the serum triglyceride levels in mice compared to untreated obese mice (pathological control) group (Figure No. 11).
Estimation of total cholesterol levels
The serum total cholesterol levels were found to be high in pathological control group when compared to other groups. The SBZN treatment significantly (P<0.01) reduced the serum total cholesterol levels in mice compared to untreated obese mice (pathological control) group (Figure No. 12).
Histopathological Studies.
The hippocampal region of the mice brain in the treatment groups was observed microscopically at 40X and compared to the pathological control and normal control for obesity induced neuronal damage or neurodegeneration. From Figure No. 13, it is clearly visible that in pathological control, the neuronal loss (gaps in the tissue) is high compared to other groups. The drug treated groups are almost near to the normal control group in terms of neuronal loss compared to obese group, which indicates the neuroprotective potential of SBZN at both the doses i.e 100mg/kg & 300mg/kg.

In nutshell. Obesity is associated with number of metabolic disorders, increased expression of pro-inflammatory markers and elevated risk for various disease including type 2 diabetes, cardiovascular disease and various types of cancer as per the method described in Haslam DW, James WP. Obesity. Lancet. 2005; 366: 1197-1209. Fundamental causes of the current obesity are associated with sedentary lifestyles, increased consumption of energy-dense foods high in saturated fats and sugars and reduced physical activity. High dietary fat induced obesity is a significant risk for cerebral oxidative stress development, neuronal inflammation, vascular dementia and AD. Recent evidences suggests that obesity apart from causing metabolic disorders also accounts for the onset of symptoms of AD in middle aged obese persons and progresses the disease by aggravating neurodegeneration in elderly patients with AD due to increased generation and deposition Aβ by cholesterol, neuroinflammation, oxidative stress, brain insulin resistance, Hyperlipidemia induced cerebral ischemia, and it also has been suggested that obesity may accelerate the normal process of aging as per method described in Tzanetakou IP, Katsilambros NL, Benetos A, Mikhailidis DP, Perrea DN. "Is obesity linked to aging": adipose tissue and the role of telomeres. Ageing Res Rev. 2012; 1: 220-229.
For the present study, the Saponin rich n-butanolic fraction was isolated from ethanolic extract of Ziziphus nummularia leaves. Saponins are secondary metabolites of plants and are reported to have lipid/cholesterol lowering, antioxidant, and anti-inflammatory and hypoglycemic properties considering this as evidence the effect of saponins on obesity induced AD onset and progression in high fat diet fed obese mice was investigated.
From the Behavioral tests i.e., rectangular maze test and Morris water maze test, it was clearly seen that there was a significant decrease in the transfer latency time in SBZN (100mg/kg & 300mg/kg) treated groups compared to obese group which indicates an improved memory in treated mice. Meanwhile locomotor activity and pole climbing tests were also conducted to determine the motor activity and learning ability of mice respectively. There was a significant difference in the values of treated groups compared to obese group.
Various biochemical parameters like blood glucose, serum triglyceride, total cholesterol and AChE enzyme levels were estimated and the SBZN at a dose of 100mg/kg & 300mg/kg had shown significant decrease in glucose, triglyceride and total cholesterol levels and increased % inhibition AChE enzyme, compared to high fat diet fed obese mice, which indicates hypoglycemic, hypolipidemic and Anti acetylcholinesterase effect of saponins.
The major antioxidants and oxidative free radical scavenging enzymes like Glutathione, SOD and Catalase plays an important role in reducing oxidative stress in brain. In this study antioxidant levels were estimated from DPPH assay and catalase enzyme levels were estimated from H2O2 scavenging assay. The high fat diet fed obese mice have shown a significant increase in the brain Malondialdehyde levels, which is a measure of lipid peroxidation and free radical generation. The antioxidant levels, catalase enzyme levels in the SBZN (100mg/kg & 300mg/kg) treated groups were significantly increased, whereas the Malondialdehyde (MDA) levels were decreased compared to pathological (obese) group.
The histopathological studies have shown prominent differences between test drug treated groups and pathological control group, which is the neurodegeneration/cell loss (gaps in the tissue) is high in obese mice compared to SBZN treated mice.
From the above results it was demonstrated that SBZN at a dose of 100mg/kg and 300mg/kg, attenuated the neurodegeneration and improved the cognitive deficits in treated mice groups compared to untreated obese mice (pathological control) group, by reversing the obesity mediated pathophysiological processes underlying AD onset and progression in mice, it clearly shows the neuroprotective potential of SBZN against obesity induced neurodegeneration.
In conclusion, the present investigation demonstrates that the saponin rich n-butanolic fraction of Z. nummularia (SBZN) got a potential to reverse the pathophysiological processes associated with obesity induced AD symptoms onset and progression (from biochemical tests), improve cognitive deficits (from behavioral tests), attenuate neurodegeneration and regenerate lost neurons (from histopathological studies), probably through lowering cholesterol levels, inhibiting lipid peroxidation, augmenting endogenous antioxidant enzymes, increasing glucose uptake and decreasing inflammatory and AChE enzyme activity in brain. In Consideration to above evidence SBZN is said to offer neuroprotection and prevent the predisposition of middle aged obese individuals to Alzheimer’s disease.
Certain specific aspects and embodiments of the present application will be explained in more detail with reference to the following examples, which are provided by way of illustration only and should not be construed as limiting the scope of the invention in any manner.
While the foregoing pages provide a detailed description of the preferred embodiments of the invention, it is to be understood that the summary, description, and examples are illustrative only of the core of the invention and non-limiting. Furthermore, as many changes can be made to the invention without departing from the scope of the invention, it is intended that all material contained herein may be interpreted as mere illustrative of the invention and not in a limiting sense.
, Claims:We Claim
1. The preparation of compositions of Saponin Rich n-Butanolic Fraction of Ziziphus nummularia Leaf Extract.
2. The composition of herbal extract as claimed in claim 1, wherein the composition comprises leaf extract of Ziziphus nummularia.
3. The composition of herbal as claimed in claim 1, wherein the composition comprises leaf extract of Ziziphus nummularia in a dose of 100-300mg/kg of body weight.
4. The composition of herbal extract as claimed in claim 1, wherein the composition comprising n-Butanolic Fraction of Ziziphus nummularia Leaf Extract has potential anti-obesity.
5. The composition of herbal extract as claimed in claim 1, wherein the composition comprising n-Butanolic Fraction of Ziziphus nummularia Leaf Extract has potential use in treatment of Alzheimer’s disease.