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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access  Full Text Article                                                                                                                                          Research Article

Phytochemical Analysis, Antioxidant and Anti Hyperlipidemic Activity of Nyctanthes arbor-tristis in Male Wistar Rats

Tanya Jain, Arpit Shrivastava *, Prateek Jain, Sunil Jain, Harshita Jain

Adina Institute of Pharmaceutical Science, Lahdara, Sagar, MP, 470001

INTRODUCTION 

A single unpaired electron causes the production of highly reactive oxygen species (ROS), which in turn causes oxidative stress and is a major factor in the pathophysiology of many physiological conditions, such as ageing, cancer, hepatic, neurological, cardiovascular, and renal disorders^1,2. Reactive oxygen radicals are produced by environmental pollutants, radiation, chemicals, poisons, deep-fried and spicy foods, as well as physical stress³. These radicals cause aberrant protein synthesis and the subsequent depletion of antioxidants within the immune system. Numerous natural antioxidant enzymes, including superoxide dismutase, catalase, and glutathione peroxidase, can deactivate free radicals and preserve normal cellular processes⁴. However, dietary antioxidants can be required if endogenous antioxidants are insufficient to sustain adequate cellular activities under elevated oxidative stress⁵. Natural diets high in phenolic and flavonoid compounds that have antioxidant activity have sparked attention in nutrition and food science in recent decades⁶. Plant secondary metabolites with an aromatic ring containing at least one hydroxyl group are known as naturally occurring phenolic and flavonoid chemicals⁷. Because of their ability to directly contribute to antioxidant action through their hydroxyl groups, phenolic substances are effective electron donors⁸. Moreover, a few of them promote the cell's natural production of antioxidant molecules⁹. Numerous studies in the literature state that phenolic substances suppress free radicals, break down peroxide, inactivate metal, scavenge oxygen, and lessen the burden of oxidative illness in biological systems¹⁰. Leafy vegetables are a great source of natural antioxidants that help shield the body from the damaging effects of free radicals¹¹. Consuming green plant vegetables, which contain phenolic and flavonoid chemicals with strong antioxidant activity, has been linked to a lower incidence of diabetes, cancer, heart disease, and neurological illnesses, according to numerous epidemiological studies¹². The primary risk factor for cardiovascular diseases (CVD) is elevated plasma concentrations of different lipids and lipoprotein fractions, which is the hallmark of the heterogeneous collection of conditions known as hyperlipidemia. Triglycerides, phospholipids, cholesterol, and cholesterol esters are some of these lipids. Large "lipoproteins" called lipids are carried by the blood and have been identified as the leading cause of death in both industrialised and developing countries. It is thought that lipid problems linked to hyperlipidemia are the cause of atherosclerotic cardiovascular disease. Reducing the risk of ischemic heart disease or the development of further cardiovascular diseases such as atherosclerosis or cerebrovascular illness is the primary goal of treatment for people with hyperlipidemia. Many adverse effects have been linked to the medications that are now on the market. Nowadays, there is a global surge in the use of complementary and alternative therapies, particularly in the use of phytochemicals. Because herbal remedies are more compatible and cause less harm than synthetic pharmaceuticals, they can increase a patient's tolerance even when used for extended periods of time^13-17. Examining the rich history of traditional medicine is crucial given the growing interest in adopting, researching, and using traditional methods based on various health care systems throughout the world. We have chosen to investigate the N. arbor-tristis in this regard. Indian folklore claims that this shrub is generally employed for religious purposes and is prized for its distinct scent¹⁸. N. arbor-tristis grows well as a shrub up to 3000 feet in height in hilly areas up to 1500 feet above sea level. The shrub reaches a height of 10 metres during its flowering season, July through October. Its leaves are tough and contain stiff, white hair. They feature an orange-colored tube that is 6 to 8 mm long, equal limbs, white lobes that are unequal, and cuneate obcordates. Their calyx is 6 to 8 mm long, and they have a glabrous corolla that is longer than 13 mm. Additionally, the plant produces opposing, simple leaves that are 6-12 cm long^19,20. The following chemical active components are found in N. arbor-tristis leaves. A few significant phytochemical components that have been identified thus far are flavanol glycosides, D-mannitol, β-sitosterol, astragaline, nicotiflorin, oleanolic acid, nyctanthic acid, ascorbic acid, and tannic acid, among others. In India, N. arbor-tristis is primarily found south of Godhavari and in the Himalayan regions. Similarly, it is extensively dispersed over Bangladesh, South-East Asia, the Indo-Pakistan peninsula, and Sub-Tropical South East Asia²¹. Key components: β-monogentiobioside of crocin-1 and crocin-3, glucose, nyctanthin, D-mannitol, tannin, β-D monoglucoside ester of α-crocetin, and carotenoids. Arbortristoside A and B, glycerides of linoleic acid, stearic acid, nyctanthic acid, 3-4 secotriterpene acid, oleic acid, lignoceric acid, and a water-soluble polymer composed of D-mannose and D-glucose are all present in the seeds of this plant. N. arbor-tristis has alkaloids and glycosides in its bark. From the plant's stem, naringenin-4-0-β-glucapyranosyl-α-xylopyranoside and β-sitosterol have been extracted. Several significant phytoconstituents, including terpenoids, anisaldehyde, phenyl acetaldehyde, and various ketones, are present in flower oil²¹. Among other significant phytoconstituents, it also includes polysaccharides, phenyl propanoid glycoside, nyctanthoside A, nyctanthic acid, friedelin, oleanolic acid, and iridoid glycosides, arbortristosides A, B, and C²². The pharmacological actions of N. arbor-tristis are extensive throughout the entire plant. For example, the leaves have anti-inflammatory, anti-fungal, antibacterial, and anti-pyretic properties. They are also used as cholagogues, diaphoretics, purgatives, and diuretics. Usually, the decoction of leaves is used to treat hepatic disorders, biliary scattering, and persistent fever.  In Ayurvedic medicine, leaf decoction is also used to treat inflammation of the joints and digestive illnesses. The other plant parts, the flowers, have strong antioxidant, diuretic, and anti-filarial properties. They are also utilised in the perfume industry. While the stem and bark have outstanding antimicrobial, antipyretic, and antioxidant properties, the seeds have antifungal, antibacterial, and immunomodulatory properties¹⁸. This study looked at the phytoconstituents found in the flowers and evaluated their antioxidant qualities. The extract from the plant has a strong ability to decrease cholesterol in diet-induced hyperlipidemia, which explains some of the plant's claimed medicinal benefits.

MATERIAL AND METHOD

Plant material

Newly blooming N. arbor-tristis gathered from Sagar, MP locality. The specimen voucher number assigned was BOT/H/03/74/234. Dr. Pradeep Tiwari, a professor in the Department of Botany at Dr. HS Gour University Sagar, (M.P.), created and authenticated the herbarium file of the plant component. Following that, the department received the herbarium file.

Chemical reagents

The following materials were obtained from SD Fine Chemicals Pvt. Ltd. in Mumbai, India: petroleum ether, ethyl acetate, methanol, sodium carbonate, potassium ferricyanide, DMSO, NaOH, ferric chloride, and trichloroacetic acid. The following ingredients were purchased from Sigma Aldrich Chemicals Pvt. Ltd. in Hyderabad, India: gallic acid, rutin, folin-ciocalteu reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH), Nitro blue tetrazolium (NBT), triton, and ascorbic acid. The remaining chemicals utilised in this investigation were procured from Hi Media Laboratories Pvt. Ltd., Lobo Chem, Ltd., SRL Pvt. Ltd., and Merck Life Sci. Private Ltd. located in Mumbai, India. Rats were used to measure their blood cholesterol levels using a commercial detection kit called Span diagnostic kit that was purchased from Surat, India. The auto analyzer Star 21 Plus Biochemistry was utilised to estimate various cholesterol levels. All other chemicals used in this study were obtained commercially and were of analytical grade and triple distilled water was used for whole experiment was generated in house.

Processing of plant

The plant material (flowers) chosen for the study were carefully cleaned under running water from the faucet and then rinsed with distilled water. They were then left to air dry at room temperature for a while. After that, the plant material was safely shade dried for three to four weeks. An electronic grinder was used to ground the dried plant material. Colour, flavour, texture, and odour of powdered plant material were noted. Dry plant material was kept for phytochemical and biological research after being sealed in an airtight container.

Extraction

Plant material was extracted for the current investigation utilising the Soxhlet apparatus and a continuous hot percolation process. N. arbor-tristis powder (250 gm) was added to a soxhlet apparatus thimble. At 60°C, soxhlation was carried out with petroleum ether serving as a non-polar solvent. After drying, the exhausted plant material (marc) was extracted again using a hydroalcoholic solvent (methanol: water; 70:30%). Each solvent's soxhlation was continued until no discernible colour change was seen in the syphon tube, and the extraction's completion was verified by the lack of any solvent residue upon evaporation. A Buchi-type rotating vacuum evaporator was used to evaporate the obtained extracts at 40°C. Weighing the dried extract, we calculated the % yield for each extract using the following formula:

Prepared extracts were observed for organoleptic characters (percentage yield, colour and odour) and were packed in air tight container and labeled till further use^23,24.

Solubility 

Dimethyl sulfoxide (DMSO), ethanol, tween 20, tween 80, normal saline (0.9 percent sodium chloride in water), and distilled water were used to examine the solubility of each extract. 

Quantitative Analysis of Phytochemicals by Spectrophotometer 

Spectrophotometer quantification of total phenolic content 

Prepare varying gallic acid concentrations (20–100μg/ml) in methanol. Prepare the test sample in 100μg/ml of methanol or a solvent with nearly the same polarity. Two millilitres of Folin-Ciocalteu Reagent (1:10 in deionized water) and four millilitres of sodium carbonate solution were added to 0.5 millilitres of various concentrations of gallic acid/test sample. With periodic shaking, incubate for 30 minutes at room temperature. Using methanol as a blank, measure the absorbance at 765 nm (because of the acquired blue hue). Create a standard curve for each gallic acid concentration and determine the regression line. Place the test sample's absorbance on the gallic acid standard curve's regression line. Gallic acid equivalent (μg/mg) or mg/gm was used to express the total phenolic content^24,25.

Spectrophotometer quantification of total flavonoid content 

Prepare varying concentrations of rutin in methanol (20–100μg/ml). Prepare the test sample in 100μg/ml of methanol or a solvent with nearly the same polarity. Blend a 0.5-ml portion of suitably diluted sample solution with 2 millilitres of distilled water, followed by 0.15 millilitres of a 5% NaNO2 solution. Add 0.15 ml of a 10% AlCl3 solution after 6 minutes, let it stand for another 6 minutes, and then mix in 2 ml of a 4% NaOH solution. Add water right away to make the mixture a final volume of 5 millilitres. Mix the mixture well and let it stand for an additional fifteen minutes. Compare the mixture's absorbance at 510 nm to a produced methanol blank. Create a standard curve for each Rutin concentration and determine the regression line. Place the test sample's absorbance on the rutin standard curve's regression line. The amount of total flavonoid content was determined and reported as mg/gm or μg/mg comparable to rutin²⁶.

Evaluation of in-vitro Antioxidant Activity 

Antioxidants are known to stop or slow down the oxidation of substrates mediated by free radicals, which helps shield the body from oxidative stress and related degenerative illnesses²⁷. Antioxidants can therefore be given externally to boost the body's own antioxidant defence mechanism. Antioxidants can function through various mechanisms and at varying degrees of oxidation. By lowering oxygen concentrations, scavenging free radicals like hydroxyl radicals to stop chain reactions from starting, or binding to free metal ions to stop metal-induced free radical formation and consequent oxidative damage, they can all lessen oxidative damage^28,29. Rich sources of phenolic and polyphenolic chemicals can be found in natural antioxidants. In order to further utilise the phytochemical content of herbal plants for the conditions linked to the disease, their antioxidant potential was assessed. The current study aimed to evaluate the extract's potential as an antioxidant and its ability to scavenge free radicals with that of ascorbic acid, a synthetic form of vitamin C, in relation to reactive oxygen species.

1- diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity

The assay method for DPPH (1, 1-diphenyl-2-picrylhydrazyl) relies on the reduction of DPPH, which is a stable free radical. The highest absorption of the odd-electron free radical DPPH occurs around 517 nm, giving rise to a purple hue. Antioxidants can cause decolorization (yellow colour) in proportion to the number of electrons captured when they react with DPPH, a stable free radical that pairs off with a hydrogen donor (such as an antioxidant that scavenges free radicals) and is reduced to the DPPHH³⁰. As a result, absorbance decreases from the DPPH radical to the DPPH-H form. The decreasing ability increases with decolorization. The most widely used model for assessing a novel drug's capacity to scavenge free radicals is this test. The technique was used to calculate the DPPH radical scavenging activity^31,32. One millilitre of a 0.1 millilitre DPPH (4 milligrammes per hundred millilitre) in methanol solution was combined with one millilitre of various extract concentrations. After giving the reaction mixture a good vortex, it was allowed to sit at room temperature for half an hour in the dark. As a reference standard, ascorbic acid was utilised, and methanol served as the control. The ability of extracts to function as antioxidants was measured by the reduction of the stable DPPH radical. Using methanolic solution as a reference solution, the UV-MINI-1240, SHIMADZU spectrophotometer was used to measure the colour change at a wavelength of 517 nm. This has to do with how well the control group absorbed the plant extracts. Every test was run in three duplicates. Despite the fact that the activity is stated as 50% inhibitory concentration (IC50), the percentage of DPPH radicals scavenged was used to determine IC50. The level of antioxidant activity increases with decreasing IC50 value. Using the following formula, the percentage inhibition of the free radical DPPH was determined: 

% inhibition = [(absorbance of control- absorbance of sample)/absorbance of control] × 100%

(DPPH) + H-A→DPPH-H + A

(Purple)                                                                                                      (Yellow)

Figure1:  Reduction of DPPH

Superoxide radical scavenging assay

The photoreduction of riboflavin produced superoxide radical (O2), which was then eliminated using the nitro blue tetrazolium dye (NBT) reduction technique. The ability of various extract fractions to scavenge super oxide radicals was reported³³ in relation to their ability to prevent the formation of formazan when nitro blue tetrazolium (NBT) was reduced photochemically. To put it succinctly, every 3 ml reaction mixture contained 0.5 ml extract/CuSO4 solution, 130 mM methionine, 60 𝜇M riboflavin, 0.5 mM EDTA, and 0.01M phosphate buffer (pH 7.8). After six minutes of exposure to fluorescent light, the absorbance of these tubes was measured at 560 nm. Superoxide radicals are produced via the nonenzymatic phenazine methosulfate-nicotinamide adenine dinucleotide (PMS-NADH) system, reducing NBT to a purple formazan. Superoxide radicals in the reaction mixture can be quenched by the plant extract and the reference component ascorbic acid, as seen by the drop in absorbance at 560 nm. As blanks, identical tubes were stored in the dark. Percent inhibition was used to express the results in relation to the control.

Superoxide radical scavenging activity (%) = [(Ao-A₁)/A_o×100]

Where A₀ is the absorbance of the control and A₁ is the absorbance of extract or the standard sample.

Animals

Wistar albino male wistar rats weighing 180-200g were kept in groups of six in controlled temperatures and humidity levels (25±2 °C, 55-65%) with a typical 12-hour light/dark cycle. Water was available at all times, along with conventional rat feed. Prior to doing the trials, the rats were given seven days to become used to the lab environment. Every experiment was conducted from 8:00 to 15:00 in a quiet room. Each series of trials had a different group of six rats. The Ministry of Environment and Forests, Government of India, New Delhi, India, established the Institutional Animal Ethics Committee (IAEC) to oversee and regulate the use of experimental animals. The IAEC granted approval for the animal experiments.

Acute toxicity studies

The Organisation for Economic Co-operation and Development (OECD) protocol for acute oral toxicity was followed³⁴. Before administering a dose, animals fast (food should be withheld overnight, but water should not be). Subsequently, the test drug is delivered and the animals are weighed. The rats that were in good health were separated into four groups. Using a curved, ball-tipped stainless steel feeding needle, the test material is given orally as a single dose. In this work, the decoction (p.o.) was administered at doses of 5, 50, 300, and 2000 mg/kg to four groups of six rats each. Three hours are spent without eating after the medication is administered. The animals are monitored for any signs of toxicity and mortality for the first two hours after treatment, then every day for up to 14 days after that, and then on occasion for up to six hours. Changes were detected in the autonomic effects (salivation, lacrimation, gauntness, and piloerection), central nervous system (gait, tremors, and convulsion), and skin and fur, eyes, and mucous membrane (nasal).

Table1: Acute toxicity study design

Clinical observation

For four hours following dosage, every animal was closely observed for any indications of toxicity. For the following 14 days, more observations are made in case there are any additional behavioural or clinical toxicity indicators. Weight changes are calculated. At the end of the test animals are weighed. The formula is used to determine LD50 values³⁵.

Dose Calculation Equation

LD₅₀ = higher dose ─ Σ (a x b)/n

Where, a = dose difference, b = animal died, n = No. of animals in each group

ED₅₀ =LD₅₀

Antihyperlipidemic Activity 

High-cholesterol diet model 

High fat diet induced hyperlipidemic model preparation of feed

A modified version of³⁶ was used to create hyperlipidemia brought on by a high-fat diet. Typical animal feed pellets were broken up into tiny pieces using a mortar and pestle, and then they were ground into a fine powder using a mixer grinder. In the mixer grinder, the remaining ingredients cholesterol 2%, citric acid 1%, sugar 40%, and coconut oil 10% were added in ascending order of quantity and thoroughly combined. The dried powder was then combined with the same amount of water each time to form little feed balls, which were subsequently refrigerated at 2°C to 8°C under plastic covers that sealed themselves. The usual group's feed was made in a similar way, with just the normal food pellets ground and water mixed in instead of any additional excipients. For every animal, this meal preparation was done once every three days. For thirty days, the animals were fed a diet heavy in fat. By assessing the rats' serum lipid and lipoprotein levels, hyperlipidemia was verified.

Experimental designs

Wistar rats weighing 180-200gm were divided into 5 groups of 6 animals each.

Group I- served +as normal control and were given only vehicle (distilled water) 

Group II- received high fat diet served as hyperlipidemic control 

Group III - received atorvastatin 10mg/kg served as standard drug 

Group IV- received (200mg/kg, p.o.) HANAT

Group V- (400mg/kg, p.o.) HANAT

Estimation of weight gain

Rats' consumption of a high-cholesterol meal and weight growth were noted during the experiment on days 0, 14, and 28 of the HANAT therapy. The approximately thirty grammes of pre-weighed food pellets were inserted into the cage's hopper. Weighing the food remained in the hopper allowed us to determine how much food each rat had eaten.

Biochemical analysis of serum

Following administration with the test medication for 29 days, the rats were kept fasting until day 30, at which point blood was extracted via retroorbital sinus puncture while the rodents were under a light anaesthesia. Serum samples from the obtained samples were utilised for a variety of biochemical assays after they were centrifuged for 10 minutes at 2000 r.p.m. Using commercial kits in accordance with manufacturer instructions, serum triglycerides (TG), total cholesterol (TC), HDL-cholesterol (HDL-C), and very low density lipoproteins were measured^37-39.

Estimation of lipids

A. Total cholesterol: An Ecoline Diagnostic Kit was used to estimate the amount of cholesterol in the serum. Detergents caused the release of cholesterol and its esters from lipoprotein. The esters are hydrolyzed by cholesterol esterase. H2O2 was produced during the enzymatic oxidation process by cholesterol oxidase. This underwent a peroxidase-catalyzed reaction with 4-aminoantipyine and phenol to become a coloured quinine mine. At 546 nm, the absorbance of the standard and the sample were measured in relation to the reagent blank value. The serum cholesterol level was reported as mg/dL.

B. Triglycerides: The Ecoline Diagnostic Kit was used to estimate the serum level of triglycerides. At 546 nm, the absorbance of the standard and the sample were measured in relation to the reagent blank value. The measurement for serum triglyceride content was mg/dL.

C. HDL cholesterol: Following the precipitation of LDL cholesterol using a phosphotungstic acid precipitating reagent, the cholesterol was extracted from the serum. Ecoline Diagnostic Kits were utilised to quantify the supernatant following centrifugation. At 546 nm, the absorbance of the standard and the sample were measured in relation to the reagent blank value. Serum HDL cholesterol is measured in milligrammes per deciliter. 

D. LDL cholesterol: LDL cholesterol was calculated by using the formula 

LDL cholesterol = Total cholesterol – [HDL cholesterol – Triglycerides/5].

LDL cholesterol level in plasma was expressed as mg/dL.

E. VLDL cholesterol: VLDL cholesterol was calculated by using the formula 

VLDL cholesterol = Total cholesterol – HDL cholesterol – Triglycerides- LDL.

Data were statically analysed as mean + SEM and expressed as just significant P<0.05 and significant P<0.01 as the case may be using one way ANOVA followed by Dunnett’s multiple comparison test^40-42.

RESULTS

In phytochemical extraction, the percentage yield of extraction is crucial for assessing the standard extraction efficiency of a given plant, different sections of the same plant, or different solvents. Using a rotary vacuum evaporator, the extracts were evaporated after the flower portion of the powdered N. arbor-tristis was separated separately using a Soxhlet device. Table 2 shows the yield of extracts made from the N. arbor-tristis flower using hydroalcoholic as a solvent. The results showed that the floral yield of N. arbor-tristis had an extract yield of 3.2% in petroleum ether and 7.8% in hydroalcoholic. Standard assays were used in qualitative phytochemical testing of extracts to investigate the presence or lack of different phytochemical elements. The hydroalcoholic extract of N. arbor-tristis was subjected to phytochemical testing. Phytochemical analysis of hydroalcoholic flower extracts from N. arbor-tristis revealed the presence of triterpenoids, steroids, alkaloids, polysaccharides, and glycosides in addition to tannins and phenolic compounds. Chemical assays were used to identify the phytoconstituents and revealed the presence of several constituents. Table 3 displays the outcomes of the N. arbor-tristis flower extract. As a result, the extracts chosen for the pharmacological investigations. Using gallic acid as a reference, the total phenolic content of the extracts was ascertained using Folin-Ciocalteu's technique. The results were given as milligrammes of the equivalent weight of gallic acid (GAE). It was discovered that the regression coefficient was R2= 0.995. The plot's intercept is 0. 063 and its slope is 0.002. Using gallic acid (20–100µg/ml) as a standard, the total phenolic contents of N. arbor-tristis extracts were determined using a regression equation based on a standard curve. It was discovered that the hydroalcoholic extract of N. arbor-tristis contained 126.000 mg GAE/gm of total phenolics. Using water as a blank, the absorbance was measured at 510 nm using a UV Spectrophotometer. Standard rutin (20–100 µg/ml) was employed.  N. arbor-tristis extracts' total flavonoid content was determined using a regression equation based on a standard curve (y=0.0012x+0.092, R2=0.9798). The plot's intercept is zero and its slope is 0.001. It was discovered that the hydroalcoholic extract of N. arbor-tristis contained 103.104 mg GAE/gm of flavonoids overall. Figures 1 and 2 in Table 4. One of the easiest and most popular techniques for evaluating the antioxidant activity of natural items is to look for DPPH radical scavenging activity. The method of DPPH radical scavenging was employed to evaluate the antioxidant activity. Ascorbic acid, the control, had antioxidant activity of 25.07µg/ml. The hydroxyl radical, singlet oxygen, and hydrogen peroxide are examples of more reactive species that are formed when superoxide anion is present and cause oxidative damage to proteins, DNA, and lipids. Consequently, one of the most crucial approaches to elucidate the mechanism of antioxidant activity is to investigate the superoxide radical scavenging activity of plant extracts. At a concentration of 1 mg/ml, ascorbic acid, the control, exhibited 36.27 µg/ml of antioxidant activity. The superoxide experiment conducted on hydroalcoholic extracts of N. arbor-tristis flower revealed antioxidant activity of 125.23µg/ml at a concentration of 1 mg/ml, as shown in Table 6.8, 6.9, and Figure 6.5, 6.6. Table 5, 6, and Figure 3, 4 show that the hydroalcoholic extract of N. arbor-tristis flower yielded 94.28µg/ml antioxidant activity at the same doses. In treated rats, there was no mortality or toxicological indication up to the upper dose of 2000 mg/kg. All 24 rats were healthy throughout the investigation and lived to see the 14-day experiment's conclusion. According to paragraphs 24 and 25 of OECD Guideline 423, animal wellbeing parameters were monitored continuously for the first two hours, then sporadically for up to six hours, and finally daily for up to fourteen days. Every group's experimental observations are methodically documented. Changes in the skin and fur, eyes, mucous membranes, respiratory and circulatory systems, autonomic and central nervous systems, soma to motor activity, and behavioural patterns are among the characteristics taken into consideration. Observations of tremor, convulsion, salivation, diarrhoea, lethargy, sleep, and coma are given particular attention. Animals given an oral hydroalcoholic extract of N. arbor-tristis at a dose of 2000 mg/kg did not exhibit any negative effects or death. Table 7. This suggests that the maximum safe dose is 2000 mg/kg. Therefore, 200 and 400 mg/kg of body weight the tenth and fifth, respectively, of the maximum acceptable dose were chosen to be studied for their in vivo anti-hyperlipidaemic effects. The hydroalcoholic extract of N. arbor-tristis had an antihyperlipidemic effect on rats fed a high-fat diet; the animals' mean body weight is displayed in Figure 5 and Table 8. Oral HANAT administration (200 mg/kg and 400 mg/kg, p.o.) in the high fat diet induce model significantly decreased serum levels of triglycerides (TG), low density lipoprotein cholesterol (LDL-C), VLDL-cholesterol, and total cholesterol (TC), but significantly increased serum levels of HDL-cholesterol when compared to the positive control group. According to this study, when HANAT was administered to rats for 30 days at doses of 200 mg/kg and 400 mg/kg, their serum lipid values were significantly (p<0.001) lower than those of the control group. When HANAT group animals were given 400 mg/kg, the results were significantly (p<0.001) different from the control group. Table 9. The triton-induced study results indicate that, in comparison to the control group, the animals' serum lipid parameters were considerably (p<0.01) reduced after receiving HANAT treatment for seven days at dose levels of 200 mg/kg and 400 mg/kg. When compared to the control group, 400 mg/kg of HANAT group animals demonstrated substantial (p<0.001) differences. Additionally, an elevated HDL level was noted at this time Table 10.

Table 2: Yield of crude extracts of N. arbor-tristis flower extract

Table3: Phytochemical screening of extract of N. arbor-tristis

Figure 1: Standard calibration curve of gallic acid for total phenolic content determination

Figure 2: Standard calibration curve of rutin for total flavonoid content

Table 4: Total phenolic and total flavonoid content of N. arbor-tristis

Table 5: DPPH radical scavenging activity of ascorbic acid and hydroalcoholic extract of N. arbor-tristis

Figure 3: DPPH radical scavenging activity of hydroalcoholic extract of N. arbor-tristis

Table 6: Superoxide scavenging activity of ascorbic acid and hydroalcoholic extract of N. arbor-tristis

Figure 4: Superoxide scavenging activity of hydroalcoholic extract of N. arbor-tristis

Table 7: Change in wellness parameters observed for N. arbor-tristis flowers extract treated wistar rats

Table 8: Effect of HANAT on weight gain in hyperlipidemia-induced rats for 4 weeks

Values are expressed as mean ± SD (n=6).Values were significant when compared with cholesterol group.*P<0.05,**P<0.01,***P<0.001(one way ANOVA followed by Dunnett test), HANAT= Hydroalcoholic extract of N. arbor-tristis flowers.

Figure 5: Effect of HANAT on weight gain in hyperlipidemia-induced rats for 4 weeks

Table 9: Effect of HANAT on lipid profile in high-cholesterol diet induced hyperlipidemia

Values were mean ±sd (n=6). Values are statistically significant at *P<0.05 and more significant at **P<0.01,***P<0.001 Vs hyperlipidemic control using one way ANOVA followed by Dunnet’s test, HANAT= Hydroalcoholic extract of N. arbor-tristis flowers.

Table 10: Effect of HANAT on lipid profile in Triton induced hyperlipidemia

Values were mean±sd (n=6). Value are statistically significant at *P<0.05andmoresignificantat**P<0.01, ***P<0.001Vs hyperlipidemic control using one way ANOVA followed by Dunnet’s test, HANAT= Hydroalcoholic extract of N. arbor-tristis flowers.

DISCUSSIONS

Hyperlipidemia is the term used to describe a high cholesterol state that has been shown to raise total cholesterol, raise the risk of atherosclerosis, and ultimately result in cardiovascular disease. Herbs are essential for the treatment of many CVDs. In India's traditional medical system and ethnomedical practices, a variety of medicinal plants and their preparations are utilised to treat hyperlipidaemia. We do not, however, have a sufficient treatment for hyperlipidaemia; instead, the majority of herbal medications expedite the lowering of cholesterol through a balanced diet. Thus, the quest for rats with elevated cholesterol levels that have anti-hyperlipidaemic activity. N. arbor-tristis, a big shrub in the Oleaceae family, is often referred to as Night Jasmine or Parijata⁴². It originated in India and is now found throughout Bangladesh, the Indo-Pak subcontinent, South East Asia, and both tropical and sub-tropical regions of the region. N. arbor-tristis is a revered, attractive, and medicinally significant shrub that is well-known throughout the nation for its fragrant white blooms. Nearly every component of this plant, including the roots, bark, leaves, flowers, seeds, and seed oil, has medicinal and industrial value. Given that it is regarded as one of the five wish-granting trees of Devaloka in Hindu mythology, garlands of flowers are produced as sacrifices to the gods. The textile industry uses orange dye made from flowers to colour cotton and silk⁴³.7.8% w/v was obtained using the soxhlet extraction process for 250 grammes of powder. Alkaloids, sugars, glycosides, tannins, phenolic chemicals, triterpenoids, and steroids are found in plant flower extracts, which may account for their antioxidant and anti-hyperlipidemic capabilities. The specific pharmacological effects were caused by the presence of several phyto-constituents such as triterpenoids, alkaloids, glucose, glycosides, tannins, and phenolic compounds. The amount of phytoconstituents is displayed in hydroalcoholic extracts. Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) assays were used to quantitatively screen crude extracts for phytochemicals by confirming the presence of flavonoids and phenolics in plant material. The measurement of the overall phenolic content, represented in milligrammes per gramme of gallic acid equivalent (GAE). It was discovered that the hydroalcoholic extract of N. arbor-tristis contained 126.000 mg GAE/gm extract of total phenolics. The extracts' overall flavonoid concentration was given as mg rutin/gm dry weight (mg rutin/gm DW). The hydroalcoholic extract of N. arbor-tristis was found to have 103.104 mg RE/gm of total flavonoids. Chemicals known as antioxidants, also known as free radical scavengers, interact with and neutralise free radicals to stop them from damaging biological systems' cells^44,45. The effectiveness of natural antioxidants as pure chemicals or as extracts from plants has been measured using in vitro techniques for assessing antioxidant activity. The results of the antioxidant screening indicate that DPPH radicals were reduced in power. The hydroalcoholic extract of N. arbor-tristis was shown to have an inhibitory concentration 50% (IC50) value of 94.28μg/ml under DPPH radical scavenging activity, which was higher than that of ascorbic acid, which was determined to be 25.07μg/ml. Regarding concentration, an activity that was dosage dependant was noted. When comparing the hydroalcoholic extract to the conventional medicine, the percentage of inhibition is lower when taking into account research on super oxide radical scavenging. Acute phase toxicological investigations on treated rats show no mortality or toxicity indicators up to a dosage limit of 2000 mg/kg. All 24 rats were healthy throughout the investigation and lived to see the 14-day experiment's conclusion. When N. arbor-tristis hydroalcoholic extract (2000 mg/kg) was given orally to animals, no negative effects or death were noted. This suggests that the maximum safe dose is 2000 mg/kg. Therefore, 200 and 400 mg/kg of body weight, or 1/10th and 1/5th of the maximal safe dose, were chosen to investigate the antihyperlipidemic activity in vivo. In this investigation, rats were given a high-fat diet as part of an experimental induction procedure. Cholesterol accumulates in endothelial cells and is transported into the bloodstream by lipoproteins. This causes the body's levels of TC, TG, LDL, and VLDL to rise and HDL to fall.  It is produced in the liver, where it is changed into bile acids and eliminated via the face. Hypercholesterolemia can lead to oxidative stress, which can lead to urinary failure. By assessing bodyweight and the activities of different lipid profiles, such as TC, TG, HDL, LDL, and VLDL, one may evaluate the function of cholesterol. When rats with hyperlipidaemia are fed a high-cholesterol diet, their body weight may rise. Increased lipoproteins that are absorbed from the intestine and processed in the liver can be transported into various tissues as dietary fat, or FFAs⁴⁶. By lowering LDL and VLDL cholesterol levels and raising HDL levels, the HANAT therapy and the medication atorvastatin dramatically reduce body weight.  When hydroalcoholic flower extracts were given orally to animals that had been forced to become hyperlipidaemic due to a high cholesterol diet, the results showed a large decrease in cholesterol, triglycerides, low density lipoproteins, and very low-density lipoproteins, as well as a significant increase in HDL cholesterol. The p value (p<0.001) indicated that the results were significant. The non-ionic detergent Triton WR 1339 causes acute hyperlipaemia by increasing cholesterol levels two to three times in a 24-hour period after treatment. Because Triton can obstruct the tissue's ability to absorb plasma lipids, it is believed that this causes increased hepatic synthesis of cholesterol, which is the mechanism underlying Triton-induced hypercholesterolemia. The triton-induced study results indicate that animals treated with HANAT at dose levels of 200 mg/kg and 400 mg/kg had considerably lower blood lipid parameters (p<0.01) than the control group; however, the 400 mg/kg of HANAT group animals showed significantly higher serum lipid parameters (p<0.001) than the control group. Additionally, a higher HDL level was noted at this time.

CONCLUSION

Antihyperlipidemic activity and possible antioxidant activity overall were revealed by the experimental and research findings pertaining to the full study of N. arbor-tristis flowers. Numerous antioxidant in vitro experiments conducted on the N. arbor-tristis flower extract show that its radical scavenging ability is well-established and roughly equivalent to that of ascorbic acid. Studies conducted on animals using hydroalcoholic extracts of N. arbor-tristis flowers showed that the extracts had a highly promising antihyperlipidemic effect, as evidenced by the considerable reduction in lipid levels observed in every animal group administered with the extracts. As a result, the animal group that received a higher extract will exhibit more activity than the group that received a lower extract or the conventional antihyperglycemic medication. Therefore, it can be concluded that the potential advantages of N. arbor-tristis hydroalcoholic extracts have been well-established and can be further utilised to demonstrate the antioxidant and antihyperlipidemic activity for regulating lipid levels and lowering the likelihood of ROS generation against free radicals. According to the research's previously reported findings, N. arbor-tristis may offer antioxidant and antihyperlipidemic properties. It is therefore advised that more research be done in order to determine the precise mechanism underlying the action of hyperlipidemia.

REFERENCES

1. Losada-Barreiro S, Bravo-Diaz C. Free radicals and polyphenols: The redox chemistry of neurodegenerative diseases. European Journal of Medicinal Chemistry. 2017; 133: 379-402. https://doi.org/10.1016/j.ejmech.2017.03.061 PMid:28415050

2. Madamanchi NR, Vendrov A, Runge MS. Oxidative stress and vascular disease. Arteriosclerosis, Thrombosis, and Vascular Biology. 2005 ;25(1):29-38. https://doi.org/10.1161/01.ATV.0000150649.39934.13 PMid:15539615

3. Agrawal S, Kulkarni GT, Sharma VN. A comparative study on the antioxidant activity of methanolic extracts of Terminalia paniculata and Madhuca longifolia. Free Radicals and Antioxidants. 2011;1(4):62-8. https://doi.org/10.5530/ax.2011.4.10

4. Kurutas EB. The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state. Nutrition journal. 2015; 15:1-22. https://doi.org/10.1186/s12937-016-0186-5 PMid:27456681 PMCid:PMC4960740

5. Rahman K. Studies on free radicals, antioxidants, and co-factors. Clinical Interventions in Aging. 2007;2(2):219-36.

6. Lee YH, Choo C, Watawana MI, Jayawardena N, Waisundara VY. An appraisal of eighteen commonly consumed edible plants as functional food based on their antioxidant and starch hydrolase inhibitory activities. Journal of the Science of Food and Agriculture. 2015;95(14):2956-64. https://doi.org/10.1002/jsfa.7039 PMid:25491037

7. Tungmunnithum D, Thongboonyou A, Pholboon A, Yangsabai A. Flavonoids and other phenolic compounds from medicinal plants for pharmaceutical and medical aspects: An overview. Medicines. 2018 ;5(3):93. https://doi.org/10.3390/medicines5030093 PMid:30149600 PMCid:PMC6165118

8. Bendary E, Francis RR, Ali HM, Sarwat MI, El Hady S. Antioxidant and structure-activity relationships (SARs) of some phenolic and anilines compounds. Annals of Agricultural Sciences. 2013; 58(2):173-81. https://doi.org/10.1016/j.aoas.2013.07.002

9. Côté J, Caillet S, Doyon G, Sylvain JF, Lacroix M. Bioactive compounds in cranberries and their biological properties. Critical Reviews in Food Science and Nutrition. 2010 ;50(7):666-79. https://doi.org/10.1080/10408390903044107 PMid:20694928

10. Aryal S, Baniya MK, Danekhu K, Kunwar P, Gurung R, Koirala N. Total phenolic content, flavonoid content and antioxidant potential of wild vegetables from Western Nepal. Plants. 2019 ;8(4):96. https://doi.org/10.3390/plants8040096 PMid:30978964 PMCid:PMC6524357

11. Yen GC, Chuang DY. Antioxidant properties of water extracts from Cassia tora L. in relation to the degree of roasting. Journal of Agricultural and Food Chemistry. 2000; 48(7):2760-5. https://doi.org/10.1021/jf991010q PMid:10898619

12. Adebooye OC, Vijayalakshmi R, Singh V. Peroxidase activity, chlorophylls and antioxidant profile of two leaf vegetables (Solanum nigrum L. and Amaranthus cruentus L.) under six pretreatment methods before cooking. International Journal of Food Science & Technology. 2008;43(1):173-8. https://doi.org/10.1111/j.1365-2621.2006.01420.x

13. Reiner Ž, Sonicki Z, Tedeschi-Reiner E. Public perceptions of cardiovascular risk factors in Croatia: the PERCRO survey. Preventive Medicine. 2010 ;51(6):494-6. https://doi.org/10.1016/j.ypmed.2010.09.015 PMid:20951724

14. Simons LA. Additive effect of plant sterol-ester margarine and cerivastatin in lowering low-density lipoprotein cholesterol in primary hypercholesterolemia. The American Journal of Cardiology. 2002;90(7):737-40. https://doi.org/10.1016/S0002-9149(02)02600-0 PMid:12356387

15. Yokozawa T, Ishida A, Cho EJ, Nakagawa T. The effects of Coptidis Rhizoma extract on a hypercholesterolemic animal model. Phytomedicine. 2003;10(1):17-22. https://doi.org/10.1078/094471103321648610 PMid:12622459

16. Balasubramanian MN, Muralidharan P, Balamurugan G. Anti hyperlipidemic activity of Pedalium murex (Linn.) fruits on high fat diet fed rats. International Journal of Pharmacology. 2008; 4(4):310-313. https://doi.org/10.3923/ijp.2008.310.313

17. Sumitra Singh SS, Sharma SK, Rajinder Mann RM. Antihyperlipidemic activity of Suaeda maritima (L.) Dumortier aerial parts in hypercholesterolemic rats. Journal of Pharmacy Research. 2012;5(3):1400-1402.

18. Gulshan B, Suri K A, Parul G. A comprehensive review on Nyctanthes arbor-tristis, International Journal of Drug Development and Research. 2015; 7(1): 183-193

19. Srivastava R, Trivedi D, Shukla G, Srivastava P. Nyctanthes arbor-tristis: A wonder Indian herbal drug needs healthcare attention. Biomedical Journal of Scientific & Technical Research. 2018; 5(3); 1-4. https://doi.org/10.26717/BJSTR.2018.05.001199

20. Bordoloi P, Devi T, Lahkar M. Evaluation of anti-inflammatory and anti-arthritic activity of ethanolic extract of leaves of Nyctanthes arbor-tristis on experimental animal models. Journal of Evolution Med. Dent. Sci.2018; 7(10):1247-1251. https://doi.org/10.14260/jemds/2018/284

21. Bordoloi P, Devi T, Lahkar M. Evaluation of anti-inflammatory and anti-arthritic activity of ethanolic extract of leaves of Nyctanthes arbor-tristis on experimental animal models. Journal of Evolution of Medical and Dental Sciences.2018; 7(10):1247-1251. https://doi.org/10.14260/jemds/2018/284

22. Priya K, Ganjewala D. Antibacterial activities and phytochemical analysis of different plant parts of Nyctanthes arbor-tristis (Linn.). Research Journal of Phytochemistry, 2007; 1(2): 61-67. https://doi.org/10.3923/rjphyto.2007.61.67

23. Khandelwal KR. Practical pharmacognosy technique and experiments. 23rd Ed. Nirali Prakashan; 2005.

24. Kokate CK. Practical pharmacognosy. 4th Ed. Vallabh Prakashan; 1994.

25. Ainsworth EA, Gillespie KM. Estimation of total phenolic content and other oxidation substrates in plant tissue using Folin-Ciocalteu reagent. Nature Protocol. 2007; 2(4): 875-877. https://doi.org/10.1038/nprot.2007.102 PMid:17446889

26. Alhakmani F, Kumar S, Khan SA. Estimation of total phenolic content, in-vitro antioxidant and anti-inflammatory activity of flowers of Moringa oleifera. Asian Pacific Journal of Tropical Biomedicine. 2013;3(8):623-7. https://doi.org/10.1016/S2221-1691(13)60126-4 PMid:23905019

27. Zhishen J, Mengcheng T, Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chemistry. 1999; 64: 555-559. https://doi.org/10.1016/S0308-8146(98)00102-2

28. Guideline Document on Acute oral Toxicity Testing, Series on Testing and Assessment No. 423. Paris: Organization for Economic Co-Operation and Development, OECD Environment, Health and Safety Publications; 1996. Available from: http//www.oecd.org/ehs.

29. Ainsworth EA, Gillespie KM. Estimation of total phenolic content and other oxidation substrates in plant tissue using Folin-Ciocalteu reagent. Nature Protocol. 2007; 2(4): 875-877. https://doi.org/10.1038/nprot.2007.102 PMid:17446889

30. Alhakmani F, Kumar S, Khan SA. Estimation of total phenolic content, in-vitro antioxidant and anti-inflammatory activity of flowers of Moringa oleifera. Asian Pacific Journal of Tropical Biomedicine. 2013;3(8):623-7. https://doi.org/10.1016/S2221-1691(13)60126-4 PMid:23905019

31. Zhishen J, Mengcheng T, Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chemistry. 1999; 64: 555-559. https://doi.org/10.1016/S0308-8146(98)00102-2

32. Aruoma OI. Free radicals, oxidative stress, and antioxidants in human health and disease. Journal of the American Oil Chemists' Society. 1998 ;75(2):199-212. https://doi.org/10.1007/s11746-998-0032-9 PMid:32287334 PMCid:PMC7101596

33. Shahidi F. Antioxidants in food and food antioxidants. Food/nahrung. 2000; 44(3):158-63. https://doi.org/10.1002/1521-3803(20000501)44:3<158::AID-FOOD158>3.0.CO;2-L PMid:10907235

34. Shahidi F. Antioxidants in plants and oleaginous seeds. Free radical in food chemistry, Nutrition and health effects. Morello MJ, Shahidi F and Ho C-T, Eds, ACS Symposium series 807. Americal Chemical Society, Washington, DC, 2002; 162-175. https://doi.org/10.1021/bk-2002-0807.ch012

35. Ismail M, Ibrar M, Iqbal Z, Hussain J, Hussain H, Ahmed M, Ejaz A, Choudhary MI. Chemical constituents and antioxidant activity of Geranium wallichianum. Records of Natural Products. 2009;3(4):193.

36. Gulçin I, Elias R, Gepdiremen A, Boyer L. Antioxidant activity of lignans from fringe tree (ChionanthusvirginicusL.). European Food Research and Technology.2006; 223: 759-767. https://doi.org/10.1007/s00217-006-0265-5

37. Jain R, Jain SK. Total Phenolic Contents and Antioxidant Activities of Some Selected Anticancer Medicinal Plants from Chhattisgarh State, India. Pharmacologyonline, 2011;2: 755-762.

38. Beauchamp C, Fridovich I. A mechanism for the production of ethylene from methional: the generation of the hydroxyl radical by xanthine oxidase. Journal of Biological Chemistry. 1970;245(18):4641-6. https://doi.org/10.1016/S0021-9258(18)62842-X PMid:5466067

39. Guideline Document on Acute oral Toxicity Testing, Series on Testing and Assessment No. 423. Paris: Organization for Economic Co-Operation and Development, OECD Environment, Health and Safety Publications; 1996. Available from: http//www.oecd.org/ehs.

40. Tomy S, Tomy S, TK UC, Kumar CS, Sjuc J. Toxicological evaluation of Diakure, an antidiabetic polyherbal formulation. International Journal of Phytomedicine. 2016:8(1):127-137

41. Sumitra Singh SS, Sharma SK, Rajinder Mann RM. Antihyperlipidemic activity of Suaeda maritima (L.) Dumortier aerial parts in hypercholesterolemic rats. Journal of Pharmacy Research. 2012; 5(3):1400-1402.

42. Sikarwar MS, Patil MB. Antihyperlipidemic activity of Salacia chinensis root extracts in triton-induced and atherogenic diet-induced hyperlipidemic rats. Indian Journal of Pharmacology. 2012;44(1):88-92. https://doi.org/10.4103/0253-7613.91875 PMid:22345877 PMCid:PMC3271547

43. Kumar S, Singh AK, Verma SK, Misra R, Seniya C. Antibacterial and phyto-chemical analysis of some medicinal plants and their efficacy on multidrug resistant bacteria. J Pure Applied Microbiology. 2013; 7:2191-204.

44. Mathur M. Herbal aphrodisiac their need, biology and status: global and regional scenario. Journal of Natural Product. 2012; 5:131-46.

45. Diplock AT, Charuleux JL, Crozier-Willi G, Kok FJ, Rice-Evans C, Roberfroid M, Stahl W, Vina-Ribes J. Functional food science and defence against reactive oxidative species. British Journal of Nutrition. 1998;80(S1): S77-112. https://doi.org/10.1079/BJN19980106 PMid:9849355

46. Van Heek M, Farley C, Compton DS, Hoos L, Alton KB, Sybertz EJ, Davis Jr HR. Comparison of the activity and disposition of the novel cholesterol absorption inhibitor, SCH58235, and its glucuronide, SCH60663. British Journal of Pharmacology. 2000;129(8):1748-54. https://doi.org/10.1038/sj.bjp.0703235 PMid:10780982 PMCid:PMC1571998