The document analyzes the phytochemical, antioxidant, and antibacterial properties of Triphala, an Ayurvedic herbal remedy. It finds that Triphala and its three constituent plants (Emblica officinalis, Terminalia chebula, and Terminalia bellerica) contain phytochemicals like carbohydrates, tannins, flavonoids, and proteins. Quantitative analysis shows the plants contain high levels of total phenols, tannins, and flavonoids. Triphala and its extracts also demonstrate strong antioxidant activity and antibacterial effects against E. coli and Bacillus subtilis. The readymade Triphala powder exhibited the highest antioxidant activity and largest inhibition zones

1. Phytochemical analysis, Antioxidant activity and
Antibacterial activity of Aqueous extract of
Triphala, a conventional herbal remedy
Submitted by
Asmita Basu
M.Sc Biotechnology, 4th Semester
Registration No.: 1330068 of 2017-2018
Roll: PG/VUOGP57/BIT-IVS No.: 036

2. Objectives
 To scientifically analysis Triphala.
 To explore the phytochemicals present within the three main
plants of Triphala.
 To explore the antioxidant and antibacterial properties present in
Triphala.

3. Introduction
 ‘Triphala’ literally means ‘three fruits’, consisting of Amalaki (Emblica
officinalis), Haritaki (Terminalia chebula), Bibhitaki (Terminalia bellerica).
 Amalaki has a cooling effect, which supports the natural functions of liver.
 Haritaki has a heating nature, which prevents bloating, gas, constipation, bile
related problems.
 Bibhitaki is particularly good for mucus problems.
 Triphala is also used in weight loss or obesity, diabetes, gastrointestinal health,
as free radical scavenger and so on.

4. Methods
Collection of Samples:
The readymade Triphala powder (Baidyanath Triphala Churn), dried Amalaki
(Emblica officinalis), Haritaki (Terminalia chebula) and Bibhitaki (Terminalia
bellerica) all were purchased from local market.
Sample Preparation:
 Sample preparation from Readymade Triphala Powder
 Sample preparation from three dried fruits- Amalaki, Haritaki and Bibhitaki
separately.

5. Methods
Qualitative Assay of Phytochemicals:
 Test for Carbohydrates:
2ml of sample + 2ml of Molisch’s reagent (followed by shaking) + 2ml of conc. Sulphuric acid
a reddish violet ring at the junction of the two layers.
 Test for Tannin:
2ml of sample + Few drops of 10% Ferric chloride solution blue or green colour.
 Test for Steroids:
2ml of sample solution + 1ml of chloroform + (2-3) drops of conc. sulphuric acid pink or red
colour.
 Test for Flavonoids:
2ml of sample solution + Few drops of lead acetate (followed by shaking) yellow precipitate.
 Test for Coumarins:
2ml sample solution + Few drops of alcoholic NaOH + conc. HCl yellow colour.

6. Methods
 Test for Alkaloids:
2ml of sample solution + 1ml Wagner’s reagent (followed by shaking) reddish-brown colour
precipitate.
 Test for Proteins:
2ml of sample solution + 20% NaOH solution an orange colour.
 Test for Cardiac glycosides:
i. Keller-killani Test- 5ml of sample solution + 2ml of glacial acetic acid + (2-3) drops of ferric
chloride solution + 1ml of conc. sulphuric acid green ring.
ii. Legals Test- 2ml of sample solution + Equal volume of sodium nitroprusside + Few drops of
NaOH pink-to-blood red precipitate.
 Test for Amino acids:
2ml of sample solution + Two drops of Ninhydrin solution purple colour.

7. Methods
Quantitative estimation of Phytochemicals:
 Determination of Total Phenol Content (TPC) and Total Tannin Content (TTC):
 Determined by using modified Folin-Ciocalteu’s method.
 Absorbance values were recorded at 720nm wavelength.
 Comparing with the standard curve of Gallic acid.
 Determination of Total Flavonoid Content (TFC):
 Determined by using the aluminum chloride assay method.
 Absorbance values were recorded at 520nm wavelength.
 Comparing with the standard curve of Quercetin.
 Determination of Total Protein Content (TPC):
 Determined by using the Lowry’s method.
 Absorbance values were recorded at 600nm.
 Comparing with the standard curve of BSA.

8. Methods
 Determination of Total Antioxidant Activity (TAA):
 Determined by using DPPH free radical scavenging assay.
 The absorbance values were taken at 520nm wavelength.
 Free radical scavenging activity expressed as percentage-
Inhibition (%) = Absorbance of control- Absorbance of sample x 100
Absorbance of control
Determination of Antibacterial activity:
 Determined by Agar-well Diffusion method.
 Bacterial strains E.Coli and Bacillus subtilis used.

9. Results
Qualitative assay of Phytochemicals:
 Table for the results of Qualitative tests of phytochemicals:
S1 = Readymade Triphala Powder
S2 = Dried Amalaki (Emblica officinalis) powder
S3 = Dried Haritaki (Terminalia chebula) powder
S4 = Dried Bibhitaki (Terminalia bellerica) powder
‘+’ = Present
‘-’ = Absent
Samples Carbohydrates Tannins Steroids Alkaloids Flavonoids Protein Cardiac
Glycosides
Coumarins Amino
acid
S1 + + - - + + + + -
S2 - + - - + + + + -
S3 - + - - + + + + -
S4 - + - - + + + + -

10. Quantitative estimation of Total Phenolic Content (TPC)
Table – OD values & conc. (mg TPC GAE/g extract) of
different sample extracts S1= Emblica officinalis, S2=
Terminalia chebula, S3= Terminalia bellerica, S4= Triphala
readymade powder
Fig – Total Phenolic Content of
different sample extracts

11. Quantitative estimation of Total Tannin Content (TTC)
Table – OD values & conc. (mg TTC GAE/g extract) of
different sample extracts S1= Emblica officinalis, S2=
Terminalia chebula, S3= Terminalia bellerica, S4= Triphala
readymade powder
Fig – Total Tannin Content of
different sample extracts

12. Quantitative estimation of Total Flavonoid Content (TFC)
Table – OD values & conc. (mg TFC QE/g extract) of
different sample extracts S1= Emblica officinalis, S2=
Terminalia chebula, S3= Terminalia bellerica, S4= Triphala
readymade powder
Fig – Total Flavonoid Content
of different sample extracts

13. Quantitative estimation of Total Protein Content
Table – OD values & conc. (mg TPC BSA/g extract) of
different sample extracts S1= Emblica officinalis,
S2= Terminalia chebula, S3= Terminalia bellerica,
S4= Triphala readymade powder
Fig – Total Protein Content of
different sample extracts

14. Quantitative estimation of Total Antioxidant Activity (TAA)
Table: OD values (520nm) and percentage of
antioxidant of different sample extracts; S1=
Emblica officinalis, S2= Terminalia chebula, S3=
Terminalia bellerica, S4= Readymade Triphala
Powder
Fig: Total Antioxidant Activity (TAA)
of different sample extracts
Samples
% of antioxidant (Mean ±
SD)
S1 44.27 ± 0.73
S2 63.02 ± 0.73
S3 71.35 ± 0.73
S4 77.6 ± 0.73

15. Fig: Determination of Antioxidant activity- C= Control, S1= Emblica
officinalis, S2= Terminalia chebula, S3= Terminalia bellerica

16. Antibacterial Activity (Agar-Well Diffusion Method)
Table: Zone of inhibition of different sample extracts; S1= Emblica officinalis, S2= Terminalia
chebula, S3= Terminalia bellerica, S4= Readymade Triphala Powder, Ab= Antibiotic, C= Control; ‘-’
denotes Not Tested or Not Detected.
Samples
Bacteria
Gram Positive (Bacillus
subtilis)
Gram Negative (E. Coli)
S1 16.66 ± 1.15 21.66 ± 0.57
S2 14.66 ± 1.15 21.66 ± 0.57
S3 12 ± 2 20.66 ± 0.57
S4 27.33 ± 1.15 20.66 ± 0.57
Ab - 28.66 ± 1.52
C - -

17. Antibacterial Activity (Agar-Well Diffusion Method)
Fig: Zone of Inhibition of different sample extract
against Bacillus subtilis
Fig: Zone of Inhibition of different sample
extract against E. Coli

18. Fig: Effects of different sample extracts against- 16. Gram Positive bacteria Bacillus
subtilis, 17. Gram Negative bacteria E. Coli
S1= Amalaki (Emblica officinalis)
S2= Haritaki (Terminalia chebula)
S3= Bibhitaki (Terminalia bellerica)
S4= Triphala Readymade Powder
Ab= Antibiotic (Chloramphenicol)
C= Control (Distilled water)

19. Conclusion
 The aqueous extracts of three dried fruits of Triphala (Emblica officinalis, Terminalia
chebula and Terminalia bellerica) as well as Readymade powder of Triphala are rich in
phytochemical constituents and antioxidants.
 Triphala samples are found to be good antibacterial agents.
 In most of the case, Readymade Triphala Powder showed the highest activity.
 A further study is required to determine the amount of dose of Triphala powder for human
consumption.

20. References
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21. Thank You…