The Preliminary Screening of Acalypha indica for Anti- Microbial and Anti-Helmintic Activity

ABOUT AUTHORS:
V. Karuna Sree*, G. Ramesh , T. Prathusha Showri, B. Sudheer Kumar, D. Chaitanya Kumar.
* Associate Professor, Department of Pharmacognosy
K.V.S.R.Siddhartha College of Pharmaceutical sciences,
Vijayawada-10, Andhra Pradesh, India.

Abstract:
The  present study  was  carried  out  to  evaluate  the anti-microbial  &  anti-helmentic  activity  of  both  alcoholic  and  water  extract  of  the  whole  plant Acalypha  indica  belongs  to  the family Euphorbiaceae . The alcoholic  and  water  extract  of  whole  plant  of  Acalypha  indica was investigated  for  its  in vitro anti-microbial  activity  by  agar  diffusion  method  &  anti-helmentic  activity  . The  crude alcoholic  and  water  extract  of  plant  inhibited  the  growth  of  both  gram-positive  bacteria   and  gram-negative  bacteria  . The gram-negative  bacteria  tested appeared  to  be  more  susceptible  to  the  extract  than  the  gram-positive  bacteria. The alcohol extract also showed significant anti-helmentic activity  against Pheretima  posthuma . The anti-microbial  &  anti-helmentic  activity  of  both  alcoholic  and  water  extract  by  comparing  with  standard  Streptomycin & Albendazole respectively.

REFERENCE ID: PHARMATUTOR-ART-1135

Introduction:
Acalypha  indica  is commonly  known  as  copper leaf , Indian  acalypha  e.t.c . It  consists  of  dried  leaves , roots and  flowers  of   Acalypha  indica  belongs to  the  family  Euphorbiaceae . The plant  grows  widely  on  the  backyards  of houses  and  through  out  the  plains  of  India . It  is  a  small  erect  herb grows  up  to  60 cms .Tribes  of  Kerala, Rajasthan  and Madhya Pradesh  use fruits: in asthma, cough, bronchitis  and  ear ache; plant  and  fruit: as  an expectorant,  laxative,  pneumonia  and rheumatism;  leaf: in  skin  diseases  like scabies. The whole plant which includes leaves, root, stem were dried and powdered by using multi mill. The powder was passed through sieve no 60, which is used for extraction. The alcohol extract was prepared by maceration, after seven days the sample was vacuum dried and evaporate the solvent to get concentrated product. The water extract was prepared by percolation using soxhlet apparatus. The prepared extracts were stored under refrigeration.

Experimental Design:
1)    Qualitative  tests
2)    Anti-microbial  activity
3)    Anti-helmintic  activity

Qualitative  tests:
a)    Test  for  flavanoids  and  glycosides
b)    Test  for  anthraquinone  glycosides
c)    Test  for  steroids  and  tri-terpenoids
                1)    Libermann-burchards  test
                2)    Nollers  test
                3)    Salkowski’s  test
d)    Test  for  reducing  sugars
e)    Test  for  tannins
f)    Test  for  saponins
g)    Test  for  alkaloids

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Anti-microbial activity:
Medium  composition:

The  sterilization  of  assay  medium  is  done  for  the  destruction  of  microbial contamination  by  the  moist  heat  with  the  help  of  autoclave. The medium composition is shown in the table 1.

Table 1: Medium composition for anti-bacterial activity

Method  of  preparation  of  test  organism  suspension:
The  test  organism  to  be  used  for  the  anti-microbial  activity  should  be 24hrs  old . The  test organism  should  be  prepared  from  the  standard  cell culture . this  cell  culture  is  streaked on  the  agar  slant  under  aseptic conditions . From  this,  the  cell  suspension  to  be  used  for  the  activity  is prepared .

The  organisms employed  here:
1)    Bacillus  subtilis
2)  Escherichia  coli

Preparation  of  the  medium: The  medium  is  prepared  by  dissolving  all  the  ingredients  in  the  water .Then  the  agar  is  added  and  dissolved  with  the  aid  of  heat . Now  this medium  is  taken  in  to  the  test  tubes  and  sterilized  in  an  autoclave

Inoculation  of  the  medium: The  medium  after  sterilization  is  taken  and  transferred  in  to  the  sterilized Petri  plates  and  inoculated  with  the  cell  suspension . They  are  placed  on  a plane  surface  to  ensure  the  proper  level  of  the  medium  on  all  sides  of the  plate . The  cup  plate  method  is  followed  here . The  cups  are  made  by a  sterile  cork  borer  gently . Care  is taken  that  the  cups  are  smoothly rounded  and  are  no  cracks  in  the  edges . All  the  cups  prepared  should have  uniform  area  and  depth .

Preparation  of  test  solutions: The  powder  of  Acalypha  indica  obtained  from  the  vaccume  drying . 200mg  of  the powder  is  taken  and  dissolved  in  4ml  of  water,  filtered And dilutions  are  made.

Water  extract    -  drug  A

Alcohol  extract -  drug  B

With  the  help  of  sterile  pipettes  the  drug  solution  should  be  placed  in the  prepared  cups  very carefully. So  that  the  drug  solution  will  not  over flow.

Refrigeration:
The  prepared  plates  are  subjected  to  refrigeration  for  1hr. so  that  the  drug solution  will  be  diffused  uniformly  from  the  cups  in  to  the  medium .

Incubation:
After  refrigeration  the  plates  are  transferred  to  the  incubator. They are incubated  for a  period  of  24hrs  during  this  incubation  period  the  bacterial growth  is  at  the  optimal  level  and  the  action  of  drug  solution  is  observed in  the  form  of  inhibition  zones . after incubation  the  inhibition  zones are developed  in  the  plates . from this the  results  are  noted  down  with  the help  of  scale .

Anti-helmintic  activity:
Samples  for  the  anti-helmentic  study  were  prepared  by  dissolving  2.5gm of  crude  extract  in  25ml of  1%  gum  acacia  solution  prepared  in  normal saline(vehicle)  to  obtain  a  stock  solution  of  100mg/ml . from  this  stock solution  different  working  dilutions  are  prepared .
The anti-helmentic  activity  was  evaluated  on  adult  Indian earthworm  Pheretima  posthuma  due  to  its  anatomical  and  physiological resemblance  with  the  intestinal  round  worm  parasites  of  human being . the activity  alcohol  and  water extract  of  whole  plant  of  Acalypha  indica  was determined . six  groups  of  equal  size  Indian  earthworms  consisting  of  three earthworms  in  each  group  were  used  for  the  study .each group  was  treated with  one  of  the  following  vehicle ( 1% gum  acacia  in  normal  saline) , albendazole ( 25mg/ml ) , alcohol  extract  ( 50mg/ml )  and  water  extract (50mg/ml ). Observations  were  made  for  the  time  taken  to  paralyze  or death  of individual  worms , paralysis  was  said  to  occur  when  the  worms  do not revive even  in  normal  saline ,death was  concluded  when  the  worms  lost their  motility  followed  with  fading  away  of  their  body  color.

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Results &discussion:

Extractive  values:

Weight  of  crude  drug      =    140gm

Weight  of  water  extract  =    14.920gm

                                                     14.920                                                                                            Water  extractive  value     =  ------------- * 100
                                                       140

                                               =    10.657% w/w

Weight  of alcohol extract  =  20.814gm

                                                    20.814                                                                                            Alcohol  extractive  value   =  ------------- * 100
                                                       140

                                               =    14.88% w/w

The alcohol and water extract values were found to be 14.88 & 10.657 respectively. The water extract is more effective over gram negative bacteria may be due to presence of alkaloids or saponins.  The alcohol extract is more effective over gram positive bacteria because of anthraquinone or steroidal glycosides. The chemical constituents present in the alcohol are also responsible for anthelmintic activity.

Conclusion:
Water  and  alcohol  extract  of  whole  plant  of  Acalypha  indica  exhibits  anti-microbial  activity  on  the  tested  organisms
                             Gram positive bacteria   (Bacillus subtilis
                             Gram negative bacteria  (Escherichia  coli)
The  alcohol  extract  of  Acalypha  indica  also  exhibits  anti-helmentic  activity. Therefore, we can  conclude  that  the  present  findings  provide  a  base  for  the  claims  made  for  the  plant .

 References:
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Table 2: Anti-helmintic activity

Table 3: Anti-microbial activity

Table 4: Qualitative tests

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