Antimitotic activity of Borassus flabellifer Seed Coat

 

Gottumukkala Krishna Mohan*, Malavika Yadav, M Sandhya Rani, Kalakotla Shanker

Centre for Pharmaceutical Sciences, IST, Jawaharlal Nehru Technological University, Hyderabad-500085, Telangana, India.

*Corresponding Author E-mail: drgkmohan@gmail.com

 

ABSTRACT:

The Antimitotic Activity of Methanolic extract of Borassus flabellifer L. (Arecaceae) seed coat (soft outer shell) was studied by using Seed Germination Assay and Allium cepa Root Tip Assay. The preliminary phytochemical screening of methanolic seed coat extract of Borassus flabellifer revealed that presence of carbohydrates, reducing sugars, coumarins, triterpenes, tannins and phenolic compounds. In Seed Germination Assay, the methanolic seed coat extract of Borassus flabellifer has significantly increased the percentage of inhibition after 24hrs, 48hrs and 72hrs treatment which was comparable with percentage of inhibition of methotrexate. At dose 50mg/ml (p<0.0001) extract has shown the inhibition of seed germination as significant as the standard drug methotrexate (0.1mg/ml)(p<0.0001). In Allium cepa root tip assay, the percentage of mitotic index was decreased significantly after 1, 3 and6 hrs of drug treatment which was comparable with the percentage mitotic index of methotrexate. At dose 150mg/ml (p<0.0001) extract has shown antimitotic activity i.e., mitotic index as significant as of methotrexate of 0.1mg/ml (p<0.0001). Thus, the Methanolic seed coat extract of Borassus flabellifer is recommended for the treatment of cancer. Further in-vivo parameters need to be done for mechanistic evaluation of drug.

 

KEYWORDS: Antimitotic activity, Borassus flabellifer, seed germination assay, Allium cepa root tip assay, Methotrexate.

 


 

INTRODUCTION:

Medicinal plants have been identified and used throughout human history. Plants have the ability to synthesize a wide variety of chemical compounds that are used to perform important biological functions and to defend against attack from predators such as insects, fungi and herbivorous animals. At least 12,000 such compounds isolated so far; a number estimated to be less than 10% of the total.1-3 Ethno botany, the study of traditional human uses of plants, is recognised as an effective way to discover future medicines.

 

In 2001, researchers identified 122 compounds used in modern medicine which were derived from “ethno medical” plant sources; 80% of these have had an ethno medical use identical or related to the current use of the active elements of the plant.4 Worldwide it is estimated that 80% of the population uses herbs; in the developing world rates could be as high as 95%. The U.S. continues to see an increase in the use of herbs. Cancer is a most common and widely known disease which is mainly characterized by the loss of mechanisms which control the processes like cell survival, proliferation and differentiation. Cancer is the second leading cause of death. Worldwide about 10 million people per year are diagnosed with cancer and more than 6 million die of the disease and over 22 million people in the world are cancer patients.26

 

Figure 1 Borassus flabellifer

 

Borassus flabellifer is a solitary, pleonanthic (does not die after flowering) palm. It can reach 30 m in height, but is typically 7-20 m. It is known as Taad in Hindi, Talam in Tamil, Karimpano in Malayalam, Tatichettu in Telugu. The plant contains several steroidal saponins, triterpenes, a polysaccharide. The fresh pulp contains vitamin A and C. The fresh sap contains vitamin B-complex. The male inflorescence contains spirostane type steroid saponins like borassosides and dioscin5.The plant also contains bitter compound called flabelliferins, these are steroidal saponins6. Borassus flabellifer is used in folk medicine and multiple purposes, such as stimulant, anti-laprotic, diuretic, antiphlogistic, antidote, wound healing, analgesic, antidiabetic etc.

 

MATERIALS AND METHODS:

Collection and Processing of Plant Material:7

Borassus flabellifer tender seeds locally termed as Thaati munjalu were obtained from the local market in summer season from Hyderabad, Telangana.

 

The seed coat ( yellowish skin or peel covering seeds) was removed. Then the seed coat is dried under shade for 2-3 days and made in to coarse powder.

 

Extraction:8-10

Extraction was done by soxhlet method. In this method normally a solid material containing some of the whole plant dried powder form material is placed inside a thimble made from thick filter paper, which is loaded into the main chamber of the Soxhlet extractor. About 100gm of plant material is subjected to soxhlet extraction by using methanol until about completion of 20-25 cycles with solvent. After extraction the solvent is removed, typically by means of a rotary evaporator, yielding the extracted compound. And their percentage yield is calculated respectively 

                                             Weight of extract

Percentage yield (%w/w)=--------------------------- X 100                                                   weight of drug taken

 

 

Extractive values: 11

The extractive values were recorded in different solvents with a view to study the distribution of various constituents of Borassus flabellifer seed coat. Accurately weighed 5.0 g of coarsely powdered air-dried material was placed in a glass-stoppered conical flask and macerated with 100 mL of the solvent for 6 hrs, shaking frequently, and then allowed to stand for 18 hrs. The mixture was filtered rapidly taking care not to lose any solvent. Twenty-five ml of the filtrate was transferred to a tared thin porcelain dish and evaporated to dryness on a water bath.

 

The residue was dried at 105°C for 6 h, cooled in a desiccator for 30 min, and weighed without delay and calculated the percentage w/w of extractive with reference to air- dried drug. 

                                             w2-w1

Extractive value ( %)  = ---------------------------- X 100

                                             weight of drug taken

 

Here , w1= weight of empty dish

          W2= weight of dish + residue

 

Fig 2- extractive values of crude drug powder with different solvents

 

PHYTOCHEMICAL SCREENING12

The process of detection of various constituents in a plant extract is known as phytochemical screening. Plants contain numerous chemical constituents that are responsible for eliciting various physiological and therapeutic responses.

 

The extracts of methanol, chloroform, ethyl acetate, n-butanol and pet.ether are then subjected to the various qualitative tests for the detection of plant constituents like alkaloids, glycosides, tannins, carbohydrates, coumarins, saponins, flavonoids, proteins etc.

 

Table 1: Drug treatment of green gram seeds in seed germination assay

Group no

Drug treatment

Concentra-tion

Treatment schedule

(hrs)

 

 

(mg/ml)

1.

Control (dis.H2O)

0

24 to 72 hrs

2.

Standard (methotrexate)

0.1mg/ml

24 to 72 hrs

3.

Methanolic extract

10mg/ml

24 to 72 hrs

4.

Methanolic extract

20mg/ml

24 to 72 hrs

5.

Methanolic extract

30mg/ml

24 to 72 hrs

6.

Methanolic extract

40mg/ml

24 to 72 hrs

7.

Methanolic extract

50mg/ml

24 to 72 hrs

 

 

 

ANTIMITOTIC ACTIVITY:

Seed Germination Assay13, 14

Seed germination assay was evaluated by using green gram seeds (Phaseolus radiatus).

 

Experimental design:

Green gram seeds were divided in to 7 groups and each group containing 4 green gram seeds.

 

Procedure:

Green gram seeds were collected from the local market and each seed weighed individually. 10 mg/ml, 20mg/ml, 30mg/ml, 40 mg/ml, 50 mg/ml concentrations of seed coat extracts were prepared. Methotrexate was used as standard drug. Distilled water was used as a control. Equal weights of seeds were added in the sample vials containing different concentrations. The vials were left at room temperature for 24hrs for imbibitions of water.

 

After 24hrs and 72hrs drug treatment dried on dry tissue paper and weighed. The time of sprouting was extended to 72hrs and photographs were taken.

                                              [ (wt D- wt E) ]

Percentage of inhibition=------------------------------X 100

                                             [ (wt D- wt M) ]

Where, wt D= Seed weight in distilled water

            wt E= Seed weight in extract sample

            wt M= Seed weight in methotrexate

 


 

 

 

  Fig 3 : Seed Germination Assay

 


 

Allium Cepa Root Tip Assay:15, 16

Experimental Design:

Allium cepa roots were divided in to 5 different groups and each group containing 3 Allium cepa roots.

 

 

 

 

 

 

Table 2: Drug Treatment of Allium cepa Roots

Group no

Drug treatment

Concentration (mg/ml)

Treatment schedule (hours)

1.

Control (dis.H2O)

0

1,3 and 6 hrs

2.

Standard (methotrexate)

0.1 mg/ml

1,3 and 6 hrs

3.

Methanolic extract

50mg/ml

1,3 and 6 hrs

4.

Methanolic extract

100mg/ml

1,3 and 6 hrs

5.

Methanolic extract

150mg/ml

1,3 and 6 hrs

 


 

 

Fig 4: Sprouting of Allium cepa Bulbs


 


 

 

 

 

Fig 5 drug treatment of roots of Allium cepa

 

 

 


Procedure:

The activity was performed as described by More et al (2006) and Williams et al (1996). Antimitotic activity was evaluated by using the meristematic cells of A. cepa roots. A. cepa bulbs were sprouted on wet stand at room temperature, when the roots were about 5 mm long the bulbs were placed on vials containing the extracts (50 mg/ml, 100mg/ml, 150 mg/ml) such that the roots were immersed in the extracts. The duration of extract treatments for each bulb was 1,3 and 6 hrs respectively. Three  bulbs were used for each extract and duration of treatment. The sprouted roots were also treated with distilled water (Control group) and methotrexate (0.1 mg/ml, Standard drug). One hour later the root tips were cut and transferred to fixing solution of 45 % acetic acid and 95 % ethanol in the ratio of 1:3 v/v (10-12 hrs) followed by warming the root tips in 1 N HCl in oven at 50ºC for 15 min and then stained with carmine stain. The slide was observed under microscope to record the number of non-dividing and dividing cells. Same procedure was repeated after 3 hrs of extract treatment. Mitotic index was calculated using the following formula.

 

                              No. of dividing cells

Mitotic index = --------------------------- X 100

                              Total no. of cells

 

 

Seed Germination Assay:

The assay resulted here that the methanolic seed coat extract of Borassus flabellifer has significantly increased the percentage of inhibition after 24hrs, 48hrs and 72hrs treatment which was comparable with percentage of inhibition of methotrexate.

 

The methanolic extract of concentration 50mg/ml (p<0.0001) has shown the antimitotic activity i.e., percentage of inhibition of seed germination as significant as the standard drug methotrexate (0.1mg/ml)(p<0.0001)

In the point of view of seed germination, the control i.e., distilled water has shown the higher seed germination compared to standard and other methanolic seed coat extracts. As the concentration of methanolic seed coat extracts was increased, the decrease in seed germination was seen. Thus, the %ge of inhibition was increased as the concentration of methanolic seed coat extracts was increased.

 


 

 

 

Fig 6 : Photographs Showing Seed Germination


 

Table 3: Values of seed germination assay

                       

 Dose

(mg/ml)           

Weights (mg) ( mean± S.E.M)after treatment

   24hrs

48hrs

72hrs

Control (dis.water)

   0

194.2± 0.4***

215.2± 0.4***

250.5± 0.4***

Standard (methotrexate)

  0.1

182.4± 0.4***

171.9± 0.5***

167.7± 0.4***

MSCE

 10

192.4± 0.3***

191.4± 0.4***

190.1± 0.6***

MSCE

 20

190.2± 0.4***

189.2± 0.5***

188.3± 0.4***

MSCE

 30

189.1± 0.6***

185.1± 0.6***

180.2± 0.4***

MSCE

 40

186.2± 0.5***

182.2± 0.3***

175.1± 0.4***

MSCE

 50

182.1± 0.4***

171.9± 0.4***

166.9± 0.4***

 

 

Fig 7 Percentage of inhibition after 72hrs

 

 

Figure 8 seed germination versus drug treatment

The * indicates p value is <0.05 which is significant, ** indicates p value is <0.01 which is very significant and *** indicates p value is <0.0001 which is extreme significant

Values are expressed as Mean ± S.E.M.

 

 

 


Methanolic seed coat extract of Borassus flabellifer

a)    Standard (methotrexate treatment alone) group was compared with control

b)   Test extracts treated groups was compared with standard (methotrexate).

Statistically analysed by one-way ANOVA followed by Dunnett’s multiple comparison test.

 

Allium cepa Root Tip Assay:

The assay resulted that the percent  mitotic index was decreased significantly after 1,3 and6 hrs of drug treatment which was comparable with the % mitotic index of methotrexate. The methanolic seed coat extract of 150mg/ml (p<0.0001) has shown antimitotic activity i.e., % mitotic index as significant as % mitotic index of Methotrexate of 0.1mg/ml (p<0.0001). The % mitotic index of methanolic seed coat extract 150mg/ml was decreased from 62% to 56.6% at 1 hr, 3hrs and 6 hrs which was compared with the methotrexate. The % mitotic index of methotrexate was found to decrease from 62% to 59.3% at 1 hr, 3hrs and 6 hrs. The mitosis was normal in the control roots. Thus, the antimitotic activity of methanolic seed coat extract of 150mg/ml was equal to the antimitotic activity shown by the standard (methotrexate). As the concentration of methanolic seed coat extract was increased, the % mitotic index was found to be decreased i.e.,increased antimitotic activity was seen.


 

 

 

 

 

Table 4 Percentage of mitotic index values of Allium cepa root tip assay

 

Dose

(mg/ml)

    % mitotic index ( mean± S.E.M) after treatment

1hr

3 hrs

6hrs

Control

   0

90.3± 0.33***

94± 0.5***

96.3± 0.8***

Standard (methotrexate)

  0.1

62± 1.15***

61.6± 0.8***

59.3± 0.6***

MSCE

  50

77.6± 0.33***

76± 0.5***

73.3± 1.2***

MSCE

 100

69.6±  0.33***

68.6± 0.3***

65.6± 0.6***

MSCE

150

62± 1.5***

57.6 ± 0.8***

56.6± 0.8***

 

 

Fig 9 Microscopic View of Alluim cepa Meristemactic Cells in Control and Standard

 

 

 

 

 

Fig 10- Microscopic view of Allium cepa Meristematic Cells in Different Concentrations of Methanolic Seed Coat Extracts.

 

 

 

 

 

Fig 11 percentage of  mitotic index versus drug treatment

The * indicates p value is <0.05 which is significant, ** indicates p value is <0.01 which is very significant and *** indicates p value is <0.0001 which is extreme significant; Values are expressed as Mean ± S.E.M.

 

 

 

 

 


Methanolic seed coat extract of Borassus flabellifer

a)    Standard (methotrexate treatment alone) group was compared with control

b)   Test extracts treated groups was compared with standard (methotrexate).

Statistically analysed by one-way ANOVA followed by Dunnett’s multiple comparison test.

 

CONCLUSION:

The phytochemical screening of different solvent extracts revealed that the more constituents were present in methanol compared to other solvents. The methanolic seed coat extract of Borassus flabellifer was found to contain carbohydrates, reducing sugars, triterpenoids, tannins and phenolic compounds. Methanolic seed coat extract of Borassus flabellifer has shown significant antimitotic activity in the seed germination assay by inhibiting the seed germination. The concentrations 10mg/ml, 20mg/ml, 30mg/ml, 40mg/ml, 50mg/ml were found to be extreme significant (p<0.0001) as comparable to the standard drug i.e., methotrexate (0.1mg/ml) (p<0.0001). In these different concentrations, the 50mg/ml has shown the more activity when and found significant . The antimitotic activity in Allium cepa root tip assay of methanolic seed coat extract of Borassus flabellifer was shown by decreasing percentage mitotic index i.e., increasing antimitotic activity. The 50mg/ml, 100mg/ml, 150mg/ml were found to be extreme significant (p<0.0001) as comparable to the standard drug i.e., methotrexate (0.1mg/ml) (p<0.0001).  Among all concentrations, the 150mg/ml has shown potent activity.

 

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Received on 26.09.2016       Modified on 10.10.2016

Accepted on 27.10.2016      ©A&V Publications All right reserved

Res.  J. Pharmacognosy and Phytochem. 2016; 8(4): 223-230.

DOI: 10.5958/0975-4385.2016.00033.9