AN APPRAISAL OF THE PHYTOCHEMICAL SCREENING OF EREMOMASTAX SPECIOSA

ABSTRACT

In this study, the phytochemical constitutes of Ereromastax speciosa leaves was investigated. These leaved as commonly used amongst traditional birth attendants in the rural regions of calabar cross river state and Akwa Ibom State, Nigeria. In this study, experienced Traditional birth attendants from the local regions in cross river and Akwa Ibom were orally interviewed about theuse of these herbs in enhancing fertility. Claims have been made by these traditional birth attendants on the efficacy of these plants for the treatment of internal heat, arresting of post-partum bleeding. Quantitative phytochemical analysis of these leaves were determined.

CHAPTER ONE

INTRODUCTION

The medicinal plants ereromastax speciosa, and Ereromastax polysperma, locally known as ikpo ikong and edem iduduot respectively amongst the efiks and ibbibios in akwa ibom and cross river state belong to the acanthaceae

family. They are tropical stout erect multibranched herbs[5], these herbs are mostly found in the tropical regions of Nigeria  and Cameroon.  They are  grown  in the  farmyards  of most  rural  dwellers for  medicinal  and ornamental purposes. Ereromastax speciosa along with its fellow genre plant  Ereromastax polysperma in combination with other herbs

have  been in  use  by traditionalist  in the  local regions  of  calabar  and  akwa ibom  to treat  women  with fertility

problems. Traditionalists by nature do not have a documented record of their herbal remedies, rather the knowledge

on the use of these herbs are passed orally from previous generations [6]. Reports have shown that  extracts from the herbs of  E. speciosa has been employed  in the treatment of dysentery,

anaemia, diarrhoea,[7] irregular, and urinary tract infection [8]. E. polysperma  plant, is  commonly called the blood tonic plant, in the treatment of diabetes, anaemia, and internal

heat the plant is mixed with eggs [9] and in the treatment of  penfigures in children[10]. 

Maternal related conditions contribute to one of the leading causes of death followed by malaria and HIV/AIDS in

developing countries [11] Women are increasingly using herbal remedies to combat fertility related conditions.[12].

In Nigeria, a survey conducted on 1200 pregnant women demonstrated that 12% used native herbs [13]. Infertility

can be the source of  a major life crises between couples [14] and this condition can lead to separation. Therefore

there is  the need  to source for  new drug  potentials in medicinal  plants that have  been in use traditionally and to

investigate their possible bioactive principles which can be used in the management of fertility issues in women. On

this note, the phytochemical analysis on the medicinal plants this study,  will contribute to knowledge in this area.

MATERIALS AND METHODS

Sample collection and preparation Ereromastax speciosa  and Ereromastax polysperma leaves were identified and authenticated in the hebarium unit of the department of botany faculty of sciences in the university of calabar, and the forestry department cross river state. Thereafter, the fresh leaves were harvested from a traditional farm at akai effa calabar  municipality. The  fresh leaves  were thoroughly  washed and  allowed to  dry under  shade. The dried leaves were blended into fine powder using a Q-link electrical blender model QBL-18L40. Two hundred and forty one (241.0g)  blended E. speciosa and one hundred and eighty point ten (180.10g) blended  E. polysperma were soaked in 2500mls of 98% ethanol as extracting solvent and allowed to stand for 72 hours at room temperature. The mixture was filtered using whatman No l filter paper to obtain a homogenous filtrate. The extract was concentrated in vacuo at a low temperature  of (37-40˚c)  using a  rotary evaporator (Model  RE52A, China).  The concentrates yielded 28.3g and 16.4g respectively. The concentrated  extracts were used for phytochemical analysis. Phytochemical analysis

Quanitative phytochemical compositions  of the leaves  were determined using  the methods described by Harbone [1], including the method of Swain[2]  for the determination of tannins , Spectrophotometric method of Brunner [3] for  the determination  of  saponnins, Folin–Ciocalteau  reagent  method [4],  for  the determination  of  polyphenols Quantitative determination of alkaloids 

This was done by the alkaline precipitation gravimetric method described by Harborne, [1]. A measured weight of the sample was dispersed in 10%  acetic acid solution in ethanol to  form a ratio of 1:10 (10%). The mixture  was allowed to  stand for 4h  at 28oC.  It was later  filtered via  what man No  42 grade of  filter paper. The  filtrate was concentrated  to  one  quarter  of  its  original  volume  by  evaporation  and  treated  with  drop  wise  addition  of conc. aqueous NH4OH until the alkaloid was precipitated. The alkaloid precipitated was received in a weighed filter paper, washed with 1% ammonia solution dried in the oven at 800C. Alkaloid content was calculated and expressed as a percentage of the weight of sample analyzed. 

Quantitative Determination of Flavonoids 

This was determined according to the method of Harborne [1]. 5gram of the sample was boiled in 50ml of 2M HCl solution for 30min under reflux. It  was allowed to cool and then  filtered through  whatman No 42 filter paper. A measured volume of the extract was treated with equal volume of ethyl acetate starting with drop.  The flavonoid precipitated was recovered by filtration using weighed filter paper. The resulting weight difference gave the weight of flavonoid in the sample.  

 Quantitative determination Tannins:  The method of Swain [2]  was used for the determination  of tannin  content. 0.2  g of  finely ground  sample was

measured into a 50 ml beaker. 20 ml of 50% methanol was added and covered with parafin and placed in a water

bath at 77-80oC for 1 h and stirred with a glass rod to prevent lumping. The extract was quantitatively filtered using

a double layered Whatman No.1 filter paper into a 100 ml volumetric flask using 50% methanol to rinse. This was

made  up  to  mark  with  distilled  water  and  thoroughly  mixed.  1  ml of  sample  extract  was  pipette  into  50  ml

volumetric flask, 20 ml distilled water, 2.5 ml Folin-Denis reagent and 10  ml of  17% Na2C03  were added  and

mixed properly. The mixture was made up to mark with distilled water, mixed well and allowed to stand for 20 min

when  a  bluish-green  colouration  developed.  Standard  Tannic  Acid  solutions  of  range  0-10  ppm  were  treated

similarly as 1 ml of sample above. The absorbances of the Tannic Acid Standard solutions as well as samples were read  after  colour  development  on  a  Spectronic  21D  Spectrophotometer  at  a  wavelength  of  760  nm.

Percentage tannin was calculated using the formula:

 Mboso O. E et al  J. Nat. Prod. Plant Resour., 2013, 3 (2):37-41 Scholars Research Library

Tannin (%) = Absorbance of sample x Average gradient x Dilution factor Weight of sample x 10,000

Quantitative determination Saponin: The Spectrophotometric method of Brunner [3] was used for saponin analysis.

1 g of finely ground sample was weighed into a 250 ml beaker and 100 ml Isobetyl alcohol was added. The mixture

was shaken on a UDY shaker for 5 h  to ensure  uniform mixing.  Thereafter, the  mixture was  filtered through  a

Whatman No.  1 filter paper  into a  100 ml beaker  and 20  ml of 40%  saturated solution of  Magnesium carbonate

added. The  mixture obtained  with saturated MgC03  was again filtered  through a Whatman No 1 filter paper  to

obtain a clear colourless solution. 1 ml of the colourless solution was pipetted into 50 ml volumetric flask and 2 ml

of 5% FeCl3 solution was added and made up to mark with distilled water. It was allowed to stand for 30 min for

blood red colour to develop. 0-10 ppm standard saponin solutions were prepared from saponin stock solution. The

standard solutions were treated similarly with  2 ml of 5% FeCl  solution as  done for  1 ml sample 3  above. The

absorbances of the sample as well as standard saponin solutions were read after colour development on a Spectronic

2lD Spectrophotometer at a wavelength of 380 nm. 

Percentage saponin was calculated using the formula:

Saponin (%) = Absorbance of sample x Average gradient x Dilution factor

                                               Weight of sample x 10,000

Quantitative Determination of Terpenes 

1gram of each of the samples was added to 10 ml of petroleum ether and allowed to extract for 15mins. The solution

was filtered and read at an absorbance of 420nm. 

3.2.2.l0 Quantitative determination of Total polyphenols

Total polyphenols were determined according to the Folin–Ciocalteau reagent method [4]. Two-hundred microlitres

of extracted sample, in triplicate, were added to 1 ml of 0.2 N Folin–Ciocalteau reagent and 0.8 ml of 7.5% sodium

carbonate solution, mixed well and allowed to stand for 30 min at room temperature. Absorption at 765 nm was read

using a Shimadzu 300 UV–Vis spectrophotometer. Quantification was based on the standard curve generated with

100– 400 mg/l of gallic acid.

 RESULTS

Quantitative estimates in percent % in the leaves of Ereromastax polysperma and ereromastax speciosa.

                                Tannin   Phenol  Flavonoids   Alkaloids  Saponins    Terpenes

Eremomastax Polysperma    

                                 15.00    55.00    977.50         652.50      975.00         1075.00

                                 ±0.00    ±0.00    ±27.50        ±2.50        ±0.00           ±25.00

Eremomastax

Speciosa   

                                 45.00    10.50    1247.50       202.50     75.00            80.00

                                 ±0.00*  ±1.50*  ±2.50*        ±2.50*     ±0.00*         ±5.00*

Values are expressed in triplicates as ±SEM @ P<0.05

DISCUSSION

The  phytochemical screening  of the  leaves  of ereromastax  plolysperma and  Ereromastax  speciosa  revealed the

presence of tannins, phenols, flavonoids, alkaloids, saponins, terpenes in varying quantities. The  theurapeutic values

of  medicinal  plants  are  attributed  to  their  phytochemical  constituents.  The  highest  constituent  of  all  the

phytochemicals in the  specie of   Ereromastax polysperma  was in  the terpenes content(1075.00±25.00). Terpenes

play  an important  role in  cellular  membrane fluidity,  as a  result  of the  triterpenes   which serve  as a  precursor

molecule  for  the  cholesterol.  Cholesterol  is  a  precursor  for  steroid  hormones  like progesterone,  estradiol,  and

testosterone. Estradiol and progesterone play a pivotal role in the  functions of the female reproductive cycle, like

ovulation, implantation and maintainance of pregnancy.  The fallopian tube  mainly functions  as a  passage for the

transport of the released egg from the ovary to the uterus, without which there will be no implantation, this function

is maintained by the healthy fallopian tubes. This therefore suggests that the plant extracts investigated  in this study

can serve as a potential drug source for the management of female fertility issues.  Terpenes, a phytochemical, is

often  used as  a potent  drug  component  against  life threatening  diseases  like  malaria  [15] heart  disease[16]and

cancer[17