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IN VITRO ANTIOXIDANT AND ANTIDIABETIC ACTIVITY OF ISOLATED FRACTION AND METHANOLIC LEAF EXTRACT OF XANTHIUM STRUMARIUM

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IN-VITRO

ANTIOXIDANT AND ANTIDIABETIC ACTIVITY OF

ISOLATED FRACTION AND METHANOLIC LEAF EXTRACT OF

XANTHIUM STRUMARIUM

Lone Umer1*, Jagrati Tripathi2, Akber Muzafar3 and Bhat Waseem4

1,3

Department of Botany S.S.L. Jain College Vidisha (M.P) India.

2

Prof. Department of Botany Unique College Bhopal (M.P) India.

4

Department of Botany Govt. Benazir Science and Commerce College (M.P) India.

ABSTRACT

The present study was aimed to investigate the effect of Isolated fraction and Methanolic extract of Xanthium strumarium leaves in normal and diabetic albino rats and its Antioxidant potential. With the aim to identify the active principle of isolated fraction responsible in lowering the blood glucose levels in experimental animals, by performing spectral analysis of fraction. The Anti-oxidant effect was determined in order to find the scavenging effect of the plant material. 9days experimental design was used to perform the experiment on Albino rats of either sex.

KEYWORDS: Methanolic extract, Xanthium strumarium, Antioxidant, Antidiabetic, Albino rats.

INTRODUCTION

Despite the presence of known antidiabetic medicine in the pharmaceutical market, diabetes and the related complications continued to be a major medical problem. Recently, some medicinal plants have been reported to be useful in diabetes worldwide and have been used empirically as antidiabetic and the most antihyper –lipidemic effects or remedies (Shukla et al, 2000). Homeostasis of glucose is maintained by the highly coordinated interaction of three physiological processes namely, insulin secretion, tissue glucose uptake and hepatic glucose production. The body keeps the supply of glucose to the cells by maintaining a constant concentration of glucose in the blood (Aguilar et al, 1998).

Volume 5, Issue 7, 950-966. Research Article ISSN 2277– 7105

*Corresponding Author

Lone Umer

Department of Botany S.S.L.

Jain College Vidisha (M.P)

India.

Article Received on 12 May 2016,

Revised on 01 June 2016, Accepted on 22 June 2016

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Diabetes mellitus is ranked seventh among the leading causes of death and is considered third when its fatal complications are taken into account. It is a metabolic disorder that is characterized by chronic hyperglycemia. It is classified into (type-1) diabetes, known as insulin-dependent diabetes mellitus and (type-2) diabetes also known as non-insulin dependent diabetes mellitus (Oh et al, 2005). The plants, which consisted of numerous active compounds, some of these compounds may act synergistically, while at times they can have antagonistic effects. The focus is now more on ethnobotany and ethnopharmacognosy in which chemists isolate and purify different sources and class of compound. (Gurub- Fakim,

2006).

As a consequence of metabolic derangement in diabetes, various complications develop, including both macro and micro vascular dysfunctions (Asulander et al., 2002) where complete cure with insulin and oral hypoglycemic agents without side effect has been challenging.

The usage of herbal medicine has amplified dramatically for various diseases amongst general people over last few years not only because of their easy accessibility without prescription, low cost and appointment to the health care specialists and more with the belief that natural remedies have less lethal effects as compared to synthetic medicines (Ashraf et al., 2011).

Medicinal plants have been described, natural ways to lower sugar in blood. They have been used more and more to complement or sometimes even replace conventional diabetic drugs (Noor et al., 2008). Studies on pharmacological effects of X.strumarium contain its antioxidant activity. (Sridharmurthy et al., 2011).

Considering that humans can be exposed to crude extracts while multiple sources, in order to compensate the effect of disease the study was designed for the betterment of human life using animal model.

MATERIALS AND METHODS

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P.G. Department of Botany Safia Science College Bhopal bearing voucher number BOT/2011/345 was preserved in botany laboratory for future reference.

The plant material was dried at room temperature, yielding 1800 gm of aerial part from the first gathering and 740gm of leaf from the second gathering.

Xanthium strumarium L. Sample Preparation

After collection, the plant material was made dust free followed by washing with distilled water. The leaves were shade dried in the laboratory at room temperature and powdered. The air-dried material was packed in air tight container for further use.

The successive Solvent extraction of crude extract of Xanthium strumarium L. leaves was done by using Soxhlet apparatus (Harborne and Baxter 1995). The Xanthium strumarium L. leaf powder was subjected to Soxhlet extraction by using methanol (Ranke, NLT 99.0% Assay GC) in 1:12 w/v ratio at 45-50°C for 48 hours till it showed dark green semisolid nature, elution of green coloured extract and dark green colour of the crude respectively. After extraction was performed, the extracts were filtered and the solvent was evaporated in rotary evaporator at 40°C and stored for further analysis, the extract yield was calculated. Further more the extract was preserved in vials of glass.

Chromatographic purification Thin layer chromatography

The methanol crude extract from the Xanthium strumarium L. leaves was subjected to thin layer chromatographic technique. TLC was produced with the aim of identifying the different compounds in a mixture and also testing purity or for separation of mixtures. Thin‐layer

chromatography (TLC) is a simple and inexpensive analytical technique that can quickly and efficiently separate quantities of less than ten micrograms of material. TLC has many applications in the organic laboratory. TLC is used for the rapid analysis of reagent and product purity, or to quickly determine the number of compounds in a mixture. Also, by comparing an unknown compound’s behavior to the behaviors of known standard

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value. The Rf value indicates the position at which a substance was located in the chromatogram.

Column Chromatography

Two methods are generally used to prepare a column, the dry method and the wet method. In column chromatography the stationary phase, a solid adsorbent is placed in a vertical glass column and the mobile phase, a liquid is added to the top which flows down through the column (By either gravity or external pressure). Column chromatography is advantageous over most other chromatographic techniques, because it can be used in both analytical and preparative applications. Not only can column chromatography be used to determine the number of components of a mixture, but it can also be used to separate and purify substantial quantities of those components for subsequent analysis.

Procedure

The methanol extract was concentrated by distilling off the solvent and evaporated to dryness. The residue (10 gm) was suspended in water, extracted successively with ethyl acetate (6×300 ml each) and then resulting solutions and were eluted with the solvent of increased polarity i.e. Non-polar - polar – highly polar.

Glass column was packed by wet filling. The slurry of adsorbent (silica gel; 60-120 mesh) was prepared by mixing the adsorbent in the chloroform and used as stationary phase. It was then poured into glass column (60cm x 4.5cm) (having sintered glass disc at its bottom) and allowed to settle. The air entrapped was removed by stirring with glass rod. A small amount of sand was kept atop the column to provide the latter a flat base. The methanolic soluble part (4.5gm) was subjected to silica gel (60-120 mesh) column (60cm x 4.5 cm) chromatography for the isolation of phytocon stituents and elution was carried out from non polar to polar solvents. The elution rate was adjusted to 10-15 ml/minute. A definite fraction was collected.

Ultraviolet–visible spectroscopy

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In-vitro anti-oxidant activity

Antioxidants are compounds that help to inhibit many oxidation reactions caused by free radicals such as singlet oxygen, superoxide, peroxyl radicals, hydroxyl radicals etc, thereby preventing or delaying the damage to the cells and tissues. As antioxidants play an important role in inhibiting and scavenging radicals, thereby providing protection to humans against infection and degenerative diseases (kumar et. al., 2007).

DPPH free Radical Scavenging assay (2, 2-diphenyl-1-picryl hydrazyl) is a stable free radical because of its spare electron delocalization over the whole molecule. The DPPH assay of Ascorbic acid and extracts was determined by the method of Pin-Der (Duh et al., (1997). Antioxidants react with DPPH, a suitable free radical, which gets reduced to DPPH-H. Consequently, the absorbance gets decreased (Braca, 2003). Using ascorbic acid as standard different concentration of ascorbic acid and extracts were prepared 200 μM solution of

DPPH in methanol was prepared and 1.5 ml of this solution was added to 1.5 ml of methanolic ascorbic acid solutions prepared in different concentrations (25, 50, 75, 100 and 125 μg/ml). The reaction mixture was vortexed thoroughly and left in the dark at room

temperature for 30min. The absorbance of the mixture was measured Spectrophotometrically against methanol (as blank) at 517nm.

The ability of the ascorbic acid and extracts to scavenge DPPH radical (Antioxidant activity) was calculated by the Equation given below,

AC-At

% Inhibition = AC × 100

Where,

% = Percentage inhibition of DPPH free radical. Ac = Absorbance of Control reaction mixture.

At = Absorbance of Test solutions at different concentrations.

Determination of antioxidant activity of isolated Fraction

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Antidiabetic activity

The Antidiabetic activity experiment was conducted on healthy experimental Albino rats Strain - Albino wistar rats.

Age - 12- 14 weeks. Sex - Either sex.

Body weight - 200 ± 300 gm.

The Albino wistar rats were obtained from the animal house, Pinnacle Biomedical Research Institute (PBRI), Bhopal India. All animal experiments were approved by Institutional Animal Ethics Committee (IAEC) of PBRI, (CPCSEA Reg. No. 1283/c/09/CPCSEA, Protocol approval No. PBRI/ 10/IAEC//PN-185) before starting the study and were conducted under the internationally accepted principles for laboratory animal use and care. All Albino wistar rats were maintained on a 12-h light/dark cycle and the temperature in the experimental animal room was maintained at 22ºC (±3ºC). They were allowed to acclimatize to laboratory conditions for a week before starting the experiment. Drinking water and food

were provided ad libitum throughout the experiment, except for the short fasting period where the drinking water was still in free access but no food supply was provided 12 h prior to treatment. The oral glucose tolerance test (OGTT) was performed for two different doses of Xanthium strumarium Linn leaf methanolic extract XSME (200 and 400 mg/kg b/w. p.o) (Bonner-Weir, 1958). Glibenclamide was dissolved in normal saline and was administered by oral Sroute at a dose of 10 mg/kg bodyweight. The Albino wistar rats divided into seven groups consisting of six animals each. The animals were then subjected to either one of the following treatments for 9 days.

Group I: Served as Normal control and received Distilled water for 9 days.

Group II: Served as Diabetic control and received Alloxan (150 mg/ kg b.w.i.p) on first day + distilled water for rest of days.

Group III: Alloxan (150 mg/kg. b.w. i.p.) on first day+ Glibenclamide 10mg/kg/day.

Group IV: Alloxan (150 mg/kg. i.p.) on first day + Methanolic extract (200mg/kg/day. Group V: Alloxan (150 mg/kg. i.p) on first day + Methanolic extract (400 mg/g/day.

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Induction of Diabetes

Diabetes was induced byAlloxan monohydrate (S.D. Fine Chemicals Ltd., Boisa). Animals were fasted for 12 hours before the experiment. The wistar injected intraperitoneally (i.p.) at a dose of 150 mg/kg body weight alloxan monohydrate freshly dissolved in sterile normal saline. Alloxan is a toxic glucose analogue, which selectively destroys insulin-producing cells in the pancreas (that is beta cells) when administered to rodents and many other animal species (Sikarwar et al., 2009).

Blood sampling, Biochemical estimation and Body weight

Blood samples were withdrawn from retro-orbital sinus under light ether (Merck, Mumbai, India) anesthesia, using heparinised capillaries and were measured immediately for glucose level determination of experiment withthe help of glucometer using strip method. During this study period the albino rats were weighed daily and their body weights were recorded with the digital electronic balance (Science Tech., India) (Priyanka et. al., 2010; Reshmi and Fatima 2001).

Statistical analysis

All the data was expressed as MEAN ± SD. Statistical analysis was carried out by using one-way ANOVA followed by Bonferroni multiple comparison tests. Values were considered statistically significant at p<0.001.

OBSERVATION AND RESULTS Thin Layer Chromatography

TLC study has shown the presence of different components present in methanolic fraction when the corresponding fractions were run in specific solvent system. Before reaching to most optimum solvent system a number of systems were employed. The TLC plate was incubated overnight at 37oC subsequently. Bioautogram developed was sprayed with 1% aqueous solution of 2, 3, 5, triphenyl tetrazolium chloride (TTC) and incubated at 37oC for four hours. The mobile phase with Toluene: Ethyl acetate (1:1) resolute compounds at 0.63,

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TLC of crude methanolic extract Xanthium strumarium leaves

Selection of mobile phase for TLC of methanol crude fraction

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Column Chromatography

The methanolic crude extract was further purified by using silica gel ‘G’ glass column and elution was carried out from non-polar to polar solvents by gradient elution method.

The methanolic fraction was chromatographed on a polyamide column with gradient system of MeOH–H2O in order to remove chlorophyll from the extract .The fractions were combined

according to their composition, yielding sub fractions I –V. Fractions other than I and II were excluded as of low yield and complexity. The isolated fraction –I was considered and selected for further study. The methanol extract (7g) was column chromatographed over Silica-gel successively eluted with CHCl3 and CHCl3-MeOH (2:1) afforded fraction A and B.

The protocol was repeated in order to attain a suitable amount of fractionate yield which was further used for the study.

Showing Separation and collection of fraction from methanolic crude extract of Xanthium strumarium leaf

Spectral analysis illuminating the results of XSFr-I which revealed best antioxidant activity, specified antidiabetic potential and through U.V analysis to determine the efficacy of isolated fraction which can be further used to determine the structure of fraction.

UV Spectroscopy

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Showing UV of Xanthium strumarium Linn

Detemination of Invitro Antioxidant activity of crude extract of Leaves of Xanthium strumarium L.

In order to determine the Antioxidant activity of methanol extract. DPPH method was employed as the method is most widely used for evaluating the antioxidant activity.

DPPH free Radical Scavenging assay

The DPPH (2, 2-diphenyl-1-picryl hydrazyl radical) analysis is one of most accurate widely employed method for evaluating antioxidant activity. Ascorbic acid was again used as standard.

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Standard curve of Ascorbic acid

Showing % Inhibition data of DPPH of free radical scavenging assay by Methanolic extract

Further the isoated fraction was subjected to antioxidant activity by using DPPH method

again.

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Graph representing regression curve of XSFr-I

Study of antidiabetic activity Acute oral toxicity (LD50)

Methanol crude extract of leaves of Xanthium strumarium L. was studied for the analysis of acute oral toxicity in order to find out the LD50 (lethal dose) for the study of antidiabetic

activity. The study was performed on rats as per OECD 423 guide lines at doses 5, 50, 300 and 2000 mg/kg. During initial hours animals were continuously observed for behavioural changes and then same procedure was followed 24 hr & 72 hr in regular intervals for 9 days. Various parameters like hyperactivity, grooming, convulsions, sedation, hypothermifa, fur colour, and number of deaths were observed. No mortality was observed up to doses of 2000mg/kg. According to the OECD guidelines for the acute toxicity, an LD50 dose of

2000mg/kg and above is categorized as unclassified and hence the drug is found to be safe (or 2000 mg/kg was considered as NOAEL (Not Observed Adverse Effect limit). As there were no lethality monitored up to dose of 2000mg/kg in the animals. Hence 1/10th and 1/5th of this NOAEL (2000mg/kg) i.e., 200mg/kg and 400mg/kg p.o. body weight were selected as dosage for in vivo antidiabetic activity and 1/40th and 1/20th of NOAEL (2000mg/kg) were calculated as 50 mg/kg b.w. and 100 mg/kg b/w. were selected as dosage for in vivo

antidiabetic activity of XSFr-I from Xanthium strumarium Linn.

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Evaluation of antidiabetic activity

The results of the effect of methanol extract and isolated Fraction (XSFr-I) which are expressed as change in blood glucose levels are shown in. The methanolic extract of leaves of

Xanthiumstrumarium and XSFr-I showed a significant (p<0.001) reduction in blood glucose levels. The blood glucose levels were reduced noticeably within 7 days of drug administration. Methanol extract doses of 200mg/kg and 400mg/kg reduced blood glucose levels when put side by side to diabetic control group. Similarly significant results were shown by the isolated XSFr- I at doses of 50mg/kg and 100mg/kg. Utmost effect was shown

by standard drug Glibenclamide by lowering blood glucose efficacy all the time. The pre-treatment blood glucose levels in Alloxan induced diabetic rats were 200-220 mg/dl. After

administration of the crude extract, the glycemic levels were found to decrease significantly from 164.395 mg/dl to 156.382 mg/dl (200 mg/kg), 176.387 mg/dl to 147.538 mg/dl (400 mg/kg) on 6th day after drug administration i.e, on 9th day and from 156.382 mg/dl to 146.542 mg/dl (200 mg/kg), 147.538 mg/dl to 128.555 mg/dl (400 mg/kg) on 9th day after drug administration. Despite the fact that the antidiabetic activity of the methanolic crude extract of leaves and its isolated fraction (XSFr-I) were significant when put side by side to diabetic control group but it was not as effectual as standard drug Glibenclamide.

Showing effect of Methanolic extract and XSFr-I on blood glucose levels (mg/dl) in Normal and Alloxan-induced diabetic rats.

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Showing effect of Methanolic extract and XSFr-I on blood glucose levels (mg/dl) in Normal and Alloxan-induced diabetic rats

Effect on body weight

Alloxan causes excellent body weight reduction in diabetic group. When compare diabetic group with the normal group, normal group shows significant blood glucose reduction. When

compare to diabetic group standard group, test groups shows blood glucose level reduction. Administration of the methanolic extract, XSFr-I and glibenclamide proved improvement in body weight gain as compared with that of the diabetic control group. During the study the

treatment with Methanolic extract and XSFr-I brought about improvement in body weights, indicating its advantageous effect in preventing loss of body weight in diabetic rats.

Showing effect of Methanolic extract and XSFr-1 on body weight in Normal and Alloxan-induced diabetic rats.

Body weight of animals.

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Showing effect of Methanolic extract and XSFr-1 on body weight in Normal and Alloxaninduced diabetic rats

DISCUSSION

Secondary metabolites have been reported to exert wide range of various biological activities which include antiviral, anti- inflammatory, antibacterial, antidiabetic, antiallergic, antitumor, treatment of neurodegenerative diseases and vasodilatory actions (Tsuchiya., 2010: Williams

et al., 2004). From the present study it was revealed that the isolated fraction was rich in secondary metabolites which aimed lowering the blood glucose levels. Antioxidants act as a defense mechanism that protects against oxidative stress or repair damaged molecules (Celiktar et al., 2007), hence the said was proved by both the Methanolic extract and isolated fraction. An abundant animal models have been developed from the past few decades for studying diabetes mellitus and testing anti-diabetic agents that consist of chemical, surgical and genetic manipulations (Srinivas et al., 2006; Etuk et al., 2010). Alloxan is a toxic source analogue, which particularly destroys insulinproducing cells in the pancreas (Etuk, 2010). After the administration of methanol crude extract of Xanthium strumarium with doses of 200mg/kg b/w and 400mg/kg b/w there was significance (p<0.001). The results are in agreement with the view that blood levels were reduced, administration of crude extract of X. strumarium Linn (Harikumar K. et al.,2012), same as proved by the isolated fraction at the dose of 50mg/kg b/w 100mg/kg b/w respectively.

ACKNOWLEDGEMENT

The Authors are highly thankful to Pinnacle Biomedical Research Institute for providing all the support during the work and also thankful to College Department for enabling us to go through the protocol that needed a platform and friendly support.

CONCLUSION

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compared to methanolic leaf extract. A large scale isolation and further spectral techniques are required to isolate and identify a particular compound responsible for lowering the blood glucose level and can be proven and used as substitute for scavenging free radicals.

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