Pharmacognostic Study, Phytochemical Screening and Neuroprotective Activity of Methanolic extract of Gynandropsis gynandra against Transient Global Ischemia induced brain damage in rats

 

D. Santhosha

Vishnu Institute of Pharmaceutical Education and Research, Narsapur, Medak. 502 313

*Corresponding Author E-mail: santhosha.d@viper.ac.in

 

ABSTRACT:

Gynandropsis gynandra is found to contain flavonoids which are proven to be potent neuroprotective agents which could cross blood brain barrier easily and treat neurotoxicity. Neurons get damaged easily because of our current lifestyle which includes usage of gadgets, smartphones etc causing memory loss, degraded cognitive thinking and other disorders like stroke. The best way to treat or prevent this is intake of flavonoids which have protective effect. The aim of the work was to assess protective effect of methanolic extract of whole herb of Gynandropsis gynandra against cerebral ischemia induced brain damage in albino wister rats. Pharmacognostic studies and phytochemical screening was carried out using standard methods. Cerebral ischemia was induced by occluding carotid artery using nylon thread followed by reperfusion. Animals were sacrificed and immediately brains were isolated and analysed for various biochemical parameter estimation, brain infarct area and histopathological studies. Biochemical estimations were measured and results were expressed in terms of Mean ± SEM by using ANNOVA software. Pharmacognostic study gave relevant information about ash values and Extractive values. Methonolic extract of Gynandropsis gynandra have proven to have protective effect. Of all the three doses, 300mg/kg dose of the drug have shown maximum protective effect when compared to other two doses.

 

KEYWORDS: Gynandropsis gynandra; ischemia; reperfusion; occlusion; carotid artery.

 

 


INTRODUCTION:

Ischemic stroke is one of the most common causes for neurological diseases and death not only in India but also in other countries. The prevalence of stroke after 55 years of age is 1 in 5 for females and 1 in 6 for males[1].

 

 

More than four-fifth part of all different kinds of strokes occur in developed and developing countries. Stroke was the cause of death in 13% which was very similar to death due to coronary artery disease (14%)[2]. The term neuroprotection indicates treatments used to protect neural tissue from cellular death or degeneration induced by deprivation of oxygen or glucose or both to the brain[3]. Cellular energy failure[4] effects cell survival in 3 ways.

 

1)   In the absence of adequate energy stores, anaerobic glycolysis is stimulated, leading to lactic acidosis.

2) Energy failure disrupts ion homeostasis. Cellular inflow of sodium and chloride with osmotically obligated water and the inflow of calcium occur.

3)   Disrupture of cell structure takes place and is due to the loss of ATP and increase in calcium concentration.

 

Gynandropsis gynandra also called as Cleome Gynandra and Ajagandha is extensively used in Ayurvedic treatment of bloating, parasitic worms, non healing wounds etc. Its seeds, leaves and roots are used for medicinal purposes. It belongs to the Family- Cleomaceae and is commonly known as ‘Hurhur’ and ‘Karaila’ in India and ‘Cat’s whiskers’ in English[5]. Flavanoids have found to be protective as they can cross blood brain barrier easily[6]. It has many pharmacological actions along with anti-oxidant activity. The flavonoids were found in a number of plants of Cleome genus either in aglycone or glycosidic form[7]. In the present study, Protective effect of Gynandropsis gynandra was assessed against transient cerebral ischemia induced brain damage or neuronal cell death in normal rats.

 

MATERIAL AND METHODS:

Plant material:

Gynandropsis gynandra was collected from Thirumala hills and authenticated by Dr. Madhava chetty with voucher number 939).

 

Acute Toxicity Studies:

The toxicity studies were conducted as per OECD Guidelines [8, 9]

Gynandropsis gynandra methonolic extract did not produce any mortality up to a dose level of 3000mg/kg body weight. (According to OECD-423). So, the three dose ranges 100mg/kg, 200mg/kg and 300mg/kg were selected for experimental studies.

 

Pharmacognostic studies:

The ash values such as total ash, acid insoluble, water soluble and sulphated ash of Gynandropsis gynandra was determined to assess the purity of the plant powder. The percentage yields of the extractives of Gynandropsis gynandra were calculated. Extractive values of the powdered samples and the qualitative microscopy on the powder characteristics were carried out as described in the Indian Pharmacopoeia [10] and Khandelwal [11].

 

Phytochemical analysis:

The extract prepared was screened for the presence of alkaloids, saponins, tannins, steroids, flavonoids, anthraquinones, cardiac glycosides and reducing sugars according to the methods available in the literature[12,13,14]

 

 

 

Powder Microscopy:[15]

Decolorising slide:

A decolorizing agent used for rendering tissues transparent and clear but leaves calcium oxalate crystals unaffected. A small quantity of powder is taken onto the slide and few drops of Chloral hydrate solution. Mix and add little of Glycerine water and apply cover slip to observe under the microscope.

 

Lignification Slide:

For second slide, add equal quantities of phloroglucinol and Conc.HCl. Mix properly and add glycerine to it. Atlast cover with coverslip and observe under the microscope.

 

Iodine Slide:

Take small quantity of Drug powder and add Iodine solution to it. Mix well, add glycerine, apply cover slip and observe under the microscope.

 

Experimental protocol[16]:

A total of 30 animals were taken, divided into five groups of six animals each. First group was treated with normal saline solution 10ml/kg b.w p.o once a day, second group was treated with normal saline 10ml/kg b.w p.o along with BCAO (Bilateral carotid artery occlusion), third group was treated with 100mg/kg b.w p.o once in a day, fourth group was treated with 200mg/kg b.w p.o once in a day and fifth group was treated with 300mg/kg b.w p.o once in a day everyday for 11 days. On the 11th day animals were fasted overnight but water was made free accessible to them. Next day animals were anaesthetized by Thiopentane sodium intraperitonially(40mg/kg).After this, animals were subjected to lay dorsally on their back, its limbs were tied to the stand, neck region was made erect by tying with a thread so that it is easy to identify and isolate carotid arteries on both sides of trachea. The isolated arteries were tied with a nylon thread to block artery so that we can stop flow of blood from heart to brain. It was left for 3hrs in this condition, meanwhile it can be left free. After 3 hrs, knot was released and again animal is left free for half an hour. The animal is sacrificed by cervical dislocation method.

 

Preparation of brain homogenate[17]:

Following decapitation, the brain was removed and washed in cooled 0.9% saline, keep on ice and subsequently blot on filter paper, then weigh and homogenize in cold phosphate buffer (0.1 M, pH 7.4) using a homogenizer. Homogenization procedure was performed as quickly as possible under completely standardized conditions. The homogenates were centrifuged at 10, 000×g for 20 min at 4°C. The following Biochemical parameters were chosen for analysis[18]

 

 

Measurement of lipid peroxidation:

TBARS, a measure of lipid peroxidation, was measured as described by Ohkawa et al.[19] According to it, 1 ml of brain homogenate was taken. To this, 0.5 ml of 30% trichloroacetic acid (TCA) and 0.5 ml of 0.8% thiobarbituric acid (TBA) reagent were added one by one. The containers were covered with aluminium foil and kept in shaking water bath for 30 minutes at 80°C. After 30 minutes, containers were taken out and kept in ice-cold water for 30 minutes. These were centrifuged at 3000 rpm for 15 minutes. The absorbance of the supernatant was measured at 540 nm at room temperature. Blank consist of 1 ml distilled water, 0.5 ml of 30% TCA, and 0.5 ml of 0.8% TBA. TBARS values were expressed as n moles malonaldehyde (MDA)/mg protein.

 

Measurement of reduced GSH:

Glutathione was measured by the method of Ellman[20]. The equal quantities of homogenate (w/v) and 10% TCA were mixed and centrifuged to separate out the proteins. Take 0.01 ml of this supernatant, add 2 ml of phosphate buffer (pH 7.4), 0.5 ml 5, 5'-dithiobisnitro benzoic acid (DTNB) and 0.4 ml of double distilled water. The mixture was mixed and the absorbance was measured at 412 nm within 15 minutes. GSH values were expressed as μ moles GSH mg protein.

 

Measurement of catalase:

Catalase activity was measured by the method of Claiborn [21]. 0.1 ml of supernatant was added to cuvette containing 1.9 ml of 50 mM phosphate buffer (pH 7). The reaction starts by the addition of 1 ml of freshly prepared 30 mM Hydrogen peroxide. The rate of decomposition of H 2 O 2 was measured spectrophotometrically at 240 nm. Catalase values were expressed as n moles of H 2 O 2 consumed/min/mg protein.

 

Measurement of superoxide dismutase:

Superoxide dismutase activity was measured by the method of Kagiyama et al[22]. One hundred μl of cytosolic supernatant was added to Tris HCl buffer (pH 8.5). The final volume of 3 ml was adjusted with the same buffer. At least 25 μl of pyrogallol was added and changes in absorbance at 420 nm were recorded at 1 minute interval for three minutes. The increase in absorbance at 420 nm after the addition of pyrogallol was inhibited by the presence of SOD.

 

Brain infarct area[17]:

Assessment of brain infarct size by TTC staining: Two brains from each group were selected for this study. Isolated brains were incubated in 2% TTC for 30min and fixation in 10% formalin for 45min at 37 ◦C. This stains living tissue pink leaving dead tissue colorless. Brain slices were made and living and dead tissues were separated to check the percentage of tissue damaged.

Histopathological studies[17]:

Two brains from each group were selected for this study. Brain tissue were fixed in 10% formalin for 24 h then washed with tap water. Paraffin bees wax tissue blocks were prepared for sectioning at 4 microns thickness by microtome. The obtained tissue sections were collected on glass slides and deparaffinised and observed under microscope.

 

Statistical Analysis:

Data was expressed as the mean ± SEM. and statistical analysis was carried out by one way ANOVA. Values of P < 0.05 were considered significant. All statistical analyses were performed and graphs were drawn using Graph Pad Prism software (version 7) computer program.

 

RESULTS:

The practical yield of the plant material is found to be 18.5% in 80%methanol. The total ash was determined and the results were tabulated in Table no.1. Total ash, Acid insoluble ash, Water soluble ash and Sulphated ash were found to be 20.7%, 14.6%, 12.5% and 16.7%. Powder microscopic characters were determined. It showed the presence of cork, wood elements, phloem fibres, Calcium oxalate crystals and trichomes. The ischemia /Reperfusion treated animals which were used for estimating biochemical parameters showed the decline in levels of superoxide dismutase, catalase and gluatatione and increase in MDA levels. The extract treated animals raised the declined levels as per the doses used. High dose drug treated rat homogenates have shown increase in levels of superoxide dismutase, catalase and glutathione and decrease in MDA levels. Brain infarct area was calculated based on the amount of white and pink matter formed by treating with TTC. The percentage of brain infarct area was increased in BCAO treated rat brain and gradually decreased in 100mg/kg, 200mg/kg and 300mg/kg of extract treated rat brains. Histopathological studies have shown cell death in BCAO treated rat brain sections when compared to normal rat brain which was recovered gradually by 100mg/kg b.w, 200mg/kg b.w and 300mg/kg b.w of plant extract.

 

Ash values:

Table 1.Percentage of different types of Ash formed (Gynandropsis gynandra)

Types of Ash

Percentage of ash

Total ash

20.7

Acid insoluble ash

14.6

Water soluble ash

12.5

Sulphated ash

16.7

 

 

Figure 1. Percentage of different types of Ash formed in a graphical manner

 

Extractive values:

Table 2. The percentage yields of the extractives of Gynandropsis gynandra

Extractives

% Yield of GG

Pet.ether

3.9

Chloroform

2.7

Methanol

18.5

Water

4.76

 

 

Figure 2.Percentage yield of Gynandropsis gynandra in different solvents in graphical manner

 

Phytochemical Screening:

Table 3. Phytochemical analysis of Gynandropsis gynandra methonolic extract

S. No

Contents

G. gynandra

1

Tannins

-

2

Alkaloids

+++

3

Proteins

+

4

Quinones

++

5

Saponin

+

6.

Terpenoid

+

7

Steroid

+

8

Flavonoids

++

9

Cardiac glycosides

+

‘+’– Present, ‘-’ -Absent

 

Powder microscopy of Gynandropsis gynandra:

Figure 3.Powder microscopic characters of Gynandropsis Gynandra in phloroglucinol and chloralhydrate solutions. a, b and c were treated with phloroglucinol and HCl. d and e were treated with chloralhydrate solution.

 

 

       

a.Trichomes b.Pholem fibres c.calcium oxlaate crystals d.wood vesseels e.cork

 

 

Biochemical parameters:

 

 

 

 

 

Figure 4. Biochemical parameters of rat brain homogenate


 

Brain Infract Area:

Table 4. Brain infarct size area in normal animals treated with Gynandropis gynandra

Group Name

Dose

(mg/kg)

Brain infact size area

Mean

SEM

A.No 1

A.No 2

A.No 3

A.No 4

A.No 5

A.No 6

Sham

-

1.1

2.15

1.1

2.29

3.84

4.45

2.49

1.39

Disease Control

-

48.35

49

54.14

57.16

53.1

60.1

53.64

4.56

Vehicle Disease control

Vehicle

51.15

53.45

59

61.15

63.65

67.9

59.38

6.28

G.gynandra

100

34.8

40.1

36

40

34

32.6

36.25

3.14

G.gynandra

200

37.8

34.5

40.15

38.3

40.4

31.15

37.05

3.59

G.gynandra

300

37

36.8

35.5

34.6

37.8

32.1

35.63

2.07

Standard

50

10.25

25.5

15.1

22

16

18

17.81

5.38

 

Histopathological Studies:

Histopathology of normal rats in Gynandropsis gynandra{GG}:

 

Figure 5.Histopathology of brain sections in Gynandropsis gynandra treated rats stained with Hematoxylin and eosin stain. Microphotograhs are observed at magnification of 10X.A-Sham-operated; B-Vehicle control; C-Ischemic control; D-100mg/kg b.w of GG; E-200mg.kg b.w of GG; F-300mg/kg b.w of GG; G-Standard-50mg/kg b.w.

 


DISCUSSION:

Plants have always proved to be helpful to mankind in one or the other ways. The most important one is in providing health. Most of the plants have medicinal properties if they are used with proper care where side effects are monitored (In case of plants where plant constituents are toxic in nature. In this study, plant extractives were analysed to estimate the percentage yield of plant in different solvents and found that, the yield was abundant in Methanol rather than chloroform, Pet.ether and water. Due to the high polarity of Methanol most of the chemical constituents of plant were dissolved in it and thus percentage yield was increased drastically than other solvents. The percentage yield of plant in methanol and water are 18.5% and 4.76% respectively. Due to higher yield in methanol, methonolic extracts were selected for further studies. Ash values were determined which could give information about the purity of the drug. It is due to the presence of high inorganic content. Powder microscopic characters were determined. Figure 3 shows different constituents identified by lignification and Iodine test.a, b and c were treated with phloroglucinol and HCl.Trichomes were stained pink indicating the presence of lignin in them.d and e were treated with chloralhydrate solution. The polyphenolics and Flavanoids which are present in the plants are proven to cross and protect brain.[23, 24, 25] In the present study, Neuroprotective effect of Gynandropsis gynandra was studied by inducing Ischemia/Reperfusion to produce ROS (Reactive oxygen species)[26, 27]. Brain is the most vulnerable organ to get damaged due to oxidative stress as neurons are rich in polyunsaturated fatty acids and levels of endogenous antioxidant enzymes in neuronal tissue are low [28, 29]. Therefore, induction of oxidative stress may lead to neuronal cell death due to I/R. When I/R is initiated, a number of events that produce ROS occur. After reperfusion, these events can set off a cascade of other biochemical and molecular sequel leading to production of ROS[30]. Fig 4 shows biochemical parameters where Catalse, superoxide simutase and Glutathione are the enzymes which are normal in control and decrease when Ischemia/Reperfusion is induced. On the other hand, MDA levels (which is the product of lipid peroxidation) will be low in normal conditions (control) and increase during Ischemia/Reperfusion treatment. Rats with extract-treatment reverse their action against Ischemia/Reperfusion treated according to the dose of the extract. At the dose of 300mg/kg almost all the biochemical parameters come to their normal condition indicating the protective activity of the extract on brain against ischemia /Reperfusion induced brain damage. In Table 4, measurement of brain infarct area showed that the normal rat brain showed complete pink indicating that the brain cells were alive. 1, 3, 5-triphenyltetrazolium chloride is a dye which when reacted with living cells turned them into pink colour whereas dead cells couldn’t take up the colour and remained colourless. When Ischemia/Reperfusion treated rat brain was made into sections it showed 53% of brain infarct area. The rats which were treated with 100mg/kg, 200mg/kg and 300mg/kg of the extract along with inducing Ischemia/Reperfusion showed slow reduction (36, 37 and 35 respectively) in the percentage of brain infarct area. This also proves the protective activity. In Figure 5, Histopathological studies have shown the rat brain sections of normal brain with no cell necrosis whereas Ischemia/Reperfusion Treated brain sections have shown major cell death. Three doses of extract treated rat brains have shown reduction in the number of cells death. Reduction rate was directly proportional to dose. As the dose increased, reduction of cell death also increased. With this, we conclude that the extract treated animals could possibly recover from the damage caused by ischemia or reperfusion which have been proved by different parameters. Gynandropsis gynandra could become a breakthrough in the area of research on neuroprotection.

 

ACKNOWLEDGEMENT:

Authors are thankful to DST, New Delhi for its financial support and Mr. Vishnu Raju, Chairman, VIPER For providing facilities for carrying out work in this institute.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 11.06.2019           Modified on 05.07.2019

Accepted on 30.07.2019          © AandV Publications All right reserved

Asian J. Res. Pharm. Sci. 2019; 9(3):156-162.

DOI: 10.5958/2231-5659.2019.00024.9