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IN VITRO STUDE

PROJECT REPORT

TITLE OF THE PROJECT : EVALUATION OF HERBOMINERAL FORMULATION T-AYU-HMTM FOR ANTIDREPANOCYTARY ACTIVITY

PRINCIPAL INVESTIGATOR : DR. M. N.SARAF

DATE :

THE BOMBAY COLLEGE OF PHARMACY
KALINA, MUMBAI-400098

TABLE OF CONTENTS:-

SR.NO CONTENTS
01 SUMMARY
02 AIM
03 MATERIALS AND METHODS
04 RESULTS
05 CONCLUSION
06 APPENDIX


1. SUMMARY:-

The herbomineral formulation T-AYU-HMTM was found to exhibit antidrepanocytary activity in Emmel’s test. At the concentration of 15ug/ml, 30ug/ml and 45ug/ml it showed 44.89%, 49.24% and 60.16% inhibition of sickling of RBC’s.


2. AIM:-
To Evaluate Antidrepanocytary (Anti sickle cell anemia) activity of T-Ayu HTTM, a Herbomineral formulation using Emmel’s test.


3. MATERIALS AND METHODS:-

Herbomineral formulation:-

Test Material:-T AYU-HMTM was supplied by Dr Atul Desai, Dhanvantri clinic, Ayurvedic healthcare centre, Sardar chowk, Vyara, Dist:-Surat, Gujarat (INDIA)

Blood samples:-
Blood samples used for evaluation of the antidrepanocytary activity of the Herbomineral formulation in this study were taken from known drepanocytary adolescent subjects from Dhanvantri clinic, Ayurvrdic Health care centre, sardar chowk, Vyara, Dist:-surat, Gujarat (INDIA).
The samples were stored at +4ºC in refrigerator.

SS BLOOD

SAMPLE NO. AGE
1 22
2 15
3 25
4 16
5 15
6 26
7 13


Biological activity:
Blood sample was put in contact with the Herbomineral formulation (T AYU-HMTM) at different concentrations prepared using saline (0.9% Sodium chloride), as per Emmel’s test procedure. In brief the method involved the following steps:

  • The blood samples and the test drug suspension were mixed in equal volumes (v\v).
  • A drop of treated\untreated blood samples was placed on slide and hermetically covered by cover glass using vaccum grease.
  • In order to ensure complete anaerobic condition, the slides were placed in vaccum desiccator and incubated at 37˚C for 24 h.
  • The number of observed erythrocytes was determined using photo microscopy under the magnification of 45X. Photo documentation of was carried out using DTV250 software.


4. RESULTS:-
The antisickling activity of the Herbomineral formulation (T AYU-HMTM) was 44.89%, 49.24%, 61.16% at concentrations of 15 µg/ml, 30 µg/ml, 45 µg/ml respectively

Table no: 01 Effect of T AYU-HMTM on Sickle cell anemia

Blood sample No.

Sickle cells inhibition (%)

Post treatment Dose concentrations (ug/ml)

15 ug/ml

30 ug/ml

45 ug/ml

1

28.24

53.86

69.68

2

63.39

65.92

68.67

3

46.95

45.69

74.72

4

18.85

37.5

67.60

5

66.60

64.29

56.44

6

28.57

3.37

12.84

7

61.62

74.07

78.16

Mean % inhibition

44.89

49.24

61.16

The Anti Drepanocytary activity of Herbomineral formulation T AYU-HMTM on blood samples No. 1 to 7 was found to be 50.59%, 65.99%, 55.78%, 41.31%, 61.44%, 14.92% and 71.28% respectively.

Blood sample no 01 showed 4-5% sickle cells before performing Emmels test. Similarly sample no 2, 3, 4, 5, 6 and & showed 6-7%, 4-5%, 3-4%, 6-7%, 2-3%and 8-9% respectively.

Percentage increase in sickle cell count post Emmel’s test in sample no. 2, 5, and 7 was 55%, 56%, and 58% respectively as compared to Blood samples No. 1, 3, 4 and 6 which exhibited 26%, 45%, 26% and 25% respectively (table no. 02). Thus we can say that the patient no 2, 5 and & 7are more susceptible to under go crisis conditions.


Table no: 03 Mean percentage inhibition of sickle cell anemia

SS BLOOD SAMPLE NO.

MEAN % INHIBITION

1

50.59

2

65.99

3

55.78

4

41.31

5

61.44

6

14.92

7

71.28


TABLE 2. Influence of T AYU-HMTM on SS Blood samples using Emmel’s test:-

 SS Blood
Samle No.

No. of Sickle cells in
SS blood
sample/100
RBC’s

No. off Sickle
cells/100 RBC’s
after Emmel’s
test

(pretreatment
to
T AYU-HMTM)

Dose
(ug/ml)

Sickle cells
after
Emmel’s test
(Post Treatment
with
T AYU-HMTM)

1

5
4

38
34

30

12

8

60

9

7

90

4

6

2

6
7

52
57

30

12

7

60

6

8

90

12

7

3

5
4

47
42

30

17

14

60

14

17

90

7

9

4

3
4

24
27

30

12

16

60

7

14

90

4

3

5

7
6

59
52

30

7

12

60

12

11

90

7

6

6

2
             3

22
27

30

10

6

60

4

7

90

5

7

7

             9
8

59
56

30

18

14

60

9

8

90

7

6



TABLE 4. Dose dependent inhibition of theHerbomineral formulation T AYU-HM on SS Blood Sample

CONCENTRATION (µg/ml)

% INHIBITION OF SICKLE CELLS

30

44.89

60

49.24

90

61.16

 

SS BLOOD SAMPLE NO.
  1 2 3 4 5 6 7 MEAN SD %
INHIBITION
CONTROL 38 52 47 24 59 22 59      
  34 57 42 27 52 27 56      
MEAN 36 54.5 44.5 25.5 55.5 24.5 57.5 42.57 14.13  
SD 2.83 3.54 3.54 2.12 4.95 3.54 2.12      
                     
TREATMENT                    
15 (ug/ml) 26.67 24.00 24.64 17.14 14.00 20.00 25.71      
  25.00 15.91 22.58 24.24 23.08 15.00 18.42      
MEAN 25.84 19.95 23.61 20.69 18.54 17.50 22.07 21.17 2.90 50.27
SD 1.18 5.72 1.45 5.02 6.42 3.54 5.16      
% SICKLE CELL
INHIBITION
28.24 63.39 46.95 18.85 66.60 28.57 61.62 44.89    
                     
                     
30 (ug/ml) 18.00 17.14 20.00 10.00 20.00 18.18 15.00      
  15.22 20.00 28.33 21.88 19.64 29.17 14.81      
MEAN 16.61 18.57 24.17 15.94 19.82 23.67 14.91 19.10 3.68 55.14
SD 1.97 2.02 5.89 8.40 0.25 7.77 0.13      
% SICKLE CELL
INHIBITION
53.86 65.92 45.69 37.5 64.29 3.37 74.07 49.24    
                     
                     
45 (ug/ml) 8.50 20.69 10.00 10.00 26.92 20.83 14.00      
  13.33 13.46 12.50 6.52 21.43 21.88 11.11      
MEAN 10.92 17.08 11.25 8.26 24.18 21.35 12.56 15.08 5.93 64.57
SD 3.42 5.11 1.77 2.46 3.89 0.74 2.04      
%SICKLE CELL
INHIBITION
69.68 68.67 74.72 67.6 56.44 12.84 78.16 61.16    

MORPHOLOGY OF DREPANOCYTES (PRETREATMENT & POST TREATMENT WITH

(T AYU-HM) USING EMMEL’S TEST (VIEWED UNDER 45X MAGNIFICATION)   

 

BLOOD SAMPLE NO. 1

Non treated SS Blood          Treated  SS Blood        T AYU-HM (15ug/ml) Treated  SS Blood   T AYU-HM(30ug/ml)   Treated SS Blood T AYU-HM (45ug/ml)

 

BLOOD SAMPLE NO. 2

Non treated SS Blood          Treated  SS Blood        T AYU-HM (15ug/ml) Treated  SS Blood   T AYU-HM(30ug/ml) Treated SS Blood T AYU-HM (45ug/ml)

 

BLOOD SAMPLE NO. 3

Non treated SS Blood          Treated  SS Blood        T AYU-HM (15ug/ml) Treated  SS Blood   T AYU-HM(30ug/ml) Treated SS Blood T AYU-HM (45ug/ml)

 

BLOOD SAMPLE NO.4

Non treated SS Blood          Treated  SS Blood        T AYU-HM (15ug/ml) Treated  SS Blood   T AYU-HM(30ug/ml) Treated SS Blood T AYU-HM (45ug/ml)

 

BLOOD SAMPLE NO. 5

Non treated SS Blood          Treated  SS Blood        T AYU-HM (15ug/ml) Treated  SS Blood   T AYU-HM(30ug/ml) Treated SS Blood T AYU-HM (45ug/ml)
BLOOD SAMPLE NO.6
Non treated SS Blood          Treated  SS Blood        T AYU-HM (15ug/ml) Treated  SS Blood   T AYU-HM(30ug/ml) Treated SS Blood T AYU-HM (45ug/ml)
BLOOD SAMPLE NO.7
  Non treated SS Blood          Treated  SS Blood        T AYU-HM (15ug/ml) Treated  SS Blood   T AYU-HM(30ug/ml) Treated SS Blood T AYU-HM (45ug/ml)

              Graph 1 and Graph 2 representing the % Sickle cell inhibition on Dose dependent manner and

               individual SS Blood sample data after treatment with Herbomineral formulation T AYU-HM                                                   

                                                                                                                       

                    Graph 1                                                      Graph 2

 

 

 

Dr. Atul Desai.
Dhanvantari Clinic
Ayurvedic Health Care Center

Sardar Chowk
Vyara-394650
099879031621
dratuldesai@rediffmail.com
info@vaidyarajayurveda.com
ayuhm4anaemia@indiatimes.com

 

 

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