INTRODUCTION:

There are numerous plants and traditional formulations available in siddha system of medicine for the treatment of many complication diseases. This has attracted a great deal of research interest in natural antioxidants, antidiabetics, antimicrobial and anti-inflammatory based drugs formulation for the prevention and treatment of complex diseases such as hepatic disorders, aging-related diseases.

Pytochemicals are the chemicals extracted from plants. These organic are classified as primary or secondary constituents, depending on their role in plant metabolism¹.

About 80% of the world's population relies solely or largely on traditional remedies for their healthcare needs.

Today, about 70,000 to 80,000 plant species are used for medicinal or aromatic purposes. In india the therapeutic use of herbs dates back to the vedic period. The Rigveda has documented about 67 medicinal plants, Yajurveda 81 species and Atharvaveda 290 species.

Medicinal plants have been used for centuries as remedies for human diseases because they contain components of therapeutic value. Medicinal plants have been identified and used throughout human history. Plants make many chemical compounds that are for biological functions, including defence against insects, fungi and herbivorous mammals. Atleast 12,000 such components have been isolated so far; a number estimated to be less than 10% of the total. The uses of herbal to treat disease is widespread in non-industrialized societies. The angle global report value of pharmaceutical plants in 2012 was over 2.2 billion².

Terminalia chebula is an important medicinal plant in indian traditional medicine in the name of “kadukkai” and it is most frequently used herbal in ayurveda.

Terminalia chebula:

It is used as a mild laxative and as an astringent wounds and abcesses. It is used as a remedy against sore throat and cough, against diarrhoea, gastroenteritis, fever, skin disease, wound infections, urinary tract infections.

MATERIALS AND METHODS:

Extraction of herbal drug – The Soxhlet apparatus:

Continuous extraction of a component from a solid mixture using methanol as a solvent. Boiling solvent vapors rise up through the larger side-arm.Condensed drops of solvent fall into the porous cup, dissolving out the desired component from a solid mixture.Condensed drops of solvent fall into the porous cup, dissolving out the desired component from a solid mixture.The solvent, containing the dissolved component, is siphoned into the boiler below.Residual solvent then drains out of the porous cup, as fresh solvent drops continue to fall into the porous cup. The sample was allowed to run in the apparatus for 48hrs at 50-60ºc. The extract was collected after the effluent has become colorless and stored for the following activity.Finally the herbal drug was extracted³.

Once the Terminalia chebula is extracted, they were subjected for Anti-microbial, Anti-Oxidant, Anti-Inflamatory and Anti-Diabetic activity. They also carried out for functional group by FTIR analysis.

Anti-Microbial activity:

Using an aseptic technique, Antibacterial activity of herbal extract was determined against pathogenic organisms such as E.coli, Salmonella, Pseudomonas, Enterococcus and Staphylococcus by Agar well diffusion method. Extract solution was prepared at 1.0µl/mL was prepared in 1% of Methanol. Using the swab, streak the Mueller-Hinton agar plate to form a bacterial lawn. To obtain uniform growth, streak the plate with the swab in one direction, rotate the plate 90° and streak the plate again in that direction. Repeat this rotation 3 times. Allow the plate to dry for approximately 5 minutes Distilled water and ampicillin acted as negative control and positive control respectively. The plates were sealed and incubated for 24 hrs at 37ºC. The zone of inhibition was observed and calculated⁴.

Anti-diabetic activity:

To different concentration of (0.5 ml, 1.0ml ,1.5ml, 2.0 ml,2.5 ml, and 3.0ml) herbal extract, 10µl of commercially available pancreatic α-amylase solution are dissolved in 1ml 20mM Sorensen buffer, pH 6.9 and 10mM NaCl. 0.1ml pancreatic α-amylase solution in 0.4% BSA is added. After prior incubation at 25ºC, the enzymatic reaction is started by addition of 1.0ml soluble starch solution. The reaction is stopped after 10min with 1.0ml of dinitrosalicylic acid (DNSA- 1g of DNSA, 30g of sodium potassium tartarate and 20 mL of 2N sodium hydroxide was added and made up to a final volume of 100 mL with distilled water) reagent. The mixture is heated in a boiling water bath for 10min, and after cooling measured at 546nm against the reagent blank. Control, representing 100% enzyme activity was prepared in a similar manner, without extract⁵.

Absorbance of sample

Parcentage of anti-dibetic activity =----------------- X 100

Absorbance of Control

Anti-Inflammatory activity:

Sample at various concentrations such as(0.5ml, 1ml, 1.5ml, 2ml, 2.5ml and 3ml) was added to the test tubes. Each test tube was made upto 4ml by using methanol. To that 400µl of Bovine serum and 5.6ml of phosphate buffer was added to all tubes and PH were adjusted by using 1N HCL. Positive control was prepared by adding 400µl of bovine serum,5.6ml phosphate buffer and 4ml of methanol. Then they, were heated in water bath for 10-15 minutes at 100ºC. OD value were measured by using spectrophotometer at 660nm and calculated by using formula^6-8.

Parcentage of Inhibition =-------------- X 100

Anti-Oxidant activity:

Samples were taken in 6 test tubes at various concentrations (such as 0.25, 0.50, 0.75, 1.00, 1.25 and 1.50ml) and made up to 3ml using methanol. 1ml of DPPH (0.3M of DPPH in methanol) solution was commonly added to all the test tubes and incubated in dark place for 30minutes. Control was prepared by adding 2ml of methanol and 1ml of DPPH solution. After incubation OD Value was measured in spectrophotometer at 517nm and calculated by using the formula^7,9,10.

Volume of control – Volume of test sample

Parcentage =-------------------------------------------- X 100

Volume of control

FT-IR analysis:

The FTIR spectrum was used to identify the functional group of the active components based on the peak value in the region of infrared radiation. The herbal powder powder of Terminalia chebula was passed into the FTIR and the functional groups of the components were separated based on its peak ratio^12-13.

GC-MS analysis:

GC-MS analysis was carried out on GC-MS-5975C [AGILENT] under the following conditions. Helium was used as the carrier gas at a constant flow rate of 1.51 ml/min and 2 μl of sample was injected. The column temperature was programmed to 70ºC with increasing temperature of 10ºC/min to 300ºC. The mass spectra were obtained through ionization energy of 70 eV in the EI mode. Total GC- MS running time was 30 min. The organic compounds were identified by comparison of mass spectra with the inbuilt libraries (NIST - 11)¹¹.

RESULTS AND DISCUSSION:

Anti-microbial activity:

Antimicrobial susceptible test was performed using various pathogen such E.coli, Enterobactericeae, staphylococcus aureus, Pseudomonas aeruginosa, salmonella typhimurium the most potent strain was successfully identified by determining by maximum zone of inhibition was found against Pseudomonas with 9nm. (Fig.1 and Table 1)

Table 1 : Anti-microbial activity with zone of inhibition

Pathogens	Test sample (µl)	Zone of inhibition (nm)
E.Coli	100	1
Staphylococcus aureus	100	1.8
Salmonella sp	100	3
Enterobacteriaceae	100	7
Pseudomonas sp	100	9

Anti-diabetic acitivty

Acarbose is a standard drug for alpha amylase inhibitor. Methanolic extract of Terminalia chebula exhibited potent alpha amylase inhibitory activity in a dose dependent manner. Ethanol extract showed highest inhibitory activity from 9% to 75% by various dependent manner (Fig 2 & Table 2).

Figure 1: Anti-microbial activity against various pathogen

Figure 2: Anti-diabetic activity

Sample (ml)	Methanol (ml)	Alpha amylase (µl)	Phosphate buffer (µl)	I N C U B A T I O N	Starch solution (µl)	I N C U B A T I O N	Iodine solution (µl)	Distilled water (ml)	OD Value 575nm	Percentage (%)
0.5	2.5	10	390		100		100	5	0.161	9
1.0	2.0	10	390		100		100	5	0.392	23
1.5	1.5	10	390		100		100	5	0.582	34
2.0	1	10	390		100		100	5	0.787	47
2.5	0.5	10	390		100		100	5	0.934	55
3.0	-	10	390		100		100	5	1.282	75
Control									1.674

Table 3 : Anti-inflammatory acitivty

Sample(ml)	Methanol (ml)	Bovine serum(µl)	Phosphate buffer (ml)	OD Value (660nm)	Percentage of inhibition (%)
0.5	3.5	400	5.60	0.169	19
1.0	3.0	400	5.60	0.232	26
1.5	2.5	400	5.60	0.592	67
2.0	2.0	400	5.60	0.896	102
2.5	1.5	400	5.60	1.208	138
3.0	1.0	400	5.60	1.533	175
Control	4.0	400	5.60	1.875

Table 4: Anti-Oxidant acitivty

Sample(ml)	Methanol (ml)	DPPH solution (ml)	INCUBATION	OD Value (517nm)	PERCENTAGE (%)
0.25	1.75	1		0.260	81
0.50	1.50	1		0.365	74
0.75	1.25	1		0.458	68
1.00	1.00	1		0.724	49
1.25	0.75	1		1.108	22
1.50	0.50	1		1.232	14
Control	2.00	1		1.433

Figure 3: Anti-inflammatory activity

Anti-Oxidant activity:

The anti-oxidant property of the beetroot juice, i.e the property of inhibiting the formation of free radicals was checked. Ascorbic acid was taken as a reference to check the percentage of anti-oxidant property of our sample. The reduction capability of DPPH radical is determined by the decrease in its absorbance at 517 nm, induced by antioxidants. The decrease in absorbance of DPPH radical is caused by antioxidants, because of the reaction between antioxidant molecules and radicals, progresses, which results in the scavenging of the radical by hydrogen donation. It is visually noticeable as a change in colour from purple to yellow. Hence, DPPH is usually used as a substrate to evaluate the antioxidative activity.

FT-IR analysis

The results of Terminalia chebula FTIR analysis confirmed the presence of amide, alkanes, carboxylic acids, aldehydes, alkenes, primary amines, aromatics, esters, ethers, alkyl halides and aliphatic amines compounds which shows major peaks at 3313.71, 2943.37, 2831.51, 1448.54, 1112.93, 1020.34 and 632.65 respectively (Fig 5 and Table 5).

Figure 4: Anti-Oxidant activity

Figure 5: FT-IR

Table 5: FT-IR with functional group

Peak value	Functional group
3313.71	Amines, Amides
2943.37	Alkanes
2831.51	Carboxylic acids
1448.54	Aromatics
1205.51	Alkyl halides
1112.93	Unknown
1020.34	Aliphatic amines
632.65	Halogen compound

GC-MS analysis:

The analyses of the volatile compounds were carried under the following conditions. Helium was used as the carrier gas at a constant flow rate of 1.51 ml/min and 2 μl of sample was injected. The mass spectra were obtained through ionization energy of 70 eV in the EI mode. Total GC- MS running time was 30 min. The preparation sample extract of NM was prepared by dissolving 1.0mg/mL in methanol (analytical grade). The GC-MS study revelas the the presence of many phytocompounds which contribute to the anti-tumor, anti-inflammatory, antibacterial and antioxidant properties. The organic compounds were identified by comparison of mass spectra with the inbuilt libraries (NIST - 11). These compounds had salubrious properties and there were more than 10 compounds such as methoxybenzyl phenylpropan, methoxymethylbiphenyl-4-yl-propan-2 and purine-6-thione, 1,7-dihydro-1-methyl (Fig 6).

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Received on 28.06.2017 Modified on 18.07.2017

Research J. Pharm. and Tech 2018; 11(5):1889-1893.

DOI: 10.5958/0974-360X.2018.00351.7