Barleria prionitis Linn (Acanthaceae) is widely distributed throughout Africa, India, Sri Lanka and tropical Asia. It is commonly known as Vajradanti, the juice of the leaf is used in cataract and fever. The dried bark is used in cough treatment and the leaves chewed to relieve toothache. The paste of the root is applied to disperse boils and glandular swellings. It exhibits several medicinal properties. The leave sare chewed to relieve toothache. Juice of the leaves is used in ulcer and fever. Paste of the roots is applied to disperse boils and glandular swellings. Leaves are also used by some tribal communities for the treatment of piles and to control irritation. Plant is also used in stiffness of limbs, enlargement of scrotum and sciatica. In recent years multiple drug resistance has developed due to indiscriminate use of existing antimicrobial drugs in the treatment of infectious diseases .Barleria prionitis, a perennial, acanthaceous, barbed, bushy medicinal plant, including in Barleria genus containing 300 species is famous for its medicinal value from ancient time.

COLLECTION, IDENTIFICATION & AUTHENTICATION

On the basis of literature survey leaves of Barleria ptionitis  Linn. (Acanthaceae) had been selected for present project. Leaves of Barleria prionitis Linn. were collected from from local market of Nanded and identified on the basis of its morphological features with the help of taxonomist. Herbarium of the plant specimen has been given for authentication to Dr. S. S. Bodke (Associate Professor & Head, Department of Botany & Horticulture, Yeshwant College, Nanded) which has been submitted to Nanded Pharmacy College, Nanded with specimen no: H-04/NPC/Pharmacology/2018-19/04 and authenticated as of Barleria ptionitis  Linn. Leaves (family: Acanthaceae) Collection, authentication, Identification, processing and storage has been done according to standard procedure for the standard procedure for the plant material (The Indian Pharmacopoeia, 2007).

 PROCESSING OF CRUDE DRUG

The collected dried leaves of plant was segregated and pulverized by mechanical grinder and the powder was passed through appropriate sieve and subjected to extraction. Powdered drug is stored in air tight container for further use.

PHARMACOGNOSTIC EVALUATION OF PLANT MATERIAL

The leaf of Barleria prionitis is dorsiventral, variable in size, 6-9.5 cm long, 2.5 - 3.5 cm wide, spines on leaf mature stem cylindrical with longitudinally arranged, greyish to light brown.

 Determination of Total Ash Content:

2 gm of the air dried crude drug weighed in a tarred silica dish and incinerated at a temperature not exceeding 4500C until free from carbon. After incineration the material was cooled and weighed. The percentage of ash value was calculated with reference to air dried drug.

Determination of Acid Insoluble Ash Value:

2 gm of the air dried crude drug weighed in a tarred silica dish and incinerated at a temperature not exceeding 4500C until free from carbon. After incineration the material was cooled and boiled with 25 ml of 2 M hydrochloric acid for 5 minutes. The insoluble matter was collected in a Gooch crucible or on ash less filter paper. The collected insoluble matter was washed with hot water, ignited and cooled in a desiccators and weighed. The percentage of acid-insoluble ash value was calculated with reference to air dried drug.

Determination of Water Soluble Ash Value:

2 gm of the air dried crude drug weighed in a tarred silica dish and incinerated at a temperature not exceeding 4500C until free from carbon. After incineration the material was cooled and boiled with 25 ml of water for 5 minutes. The insoluble matter was collected in a Gooch crucible or on ash less filter paper. The collected insoluble matter was washed with hot water, ignited and cooled in desiccators and weighed. The percentage of water soluble and water-insoluble ash value was calculated with reference to air dried drug.

Determination of Loss on Drying (LOD):

Glass-stopper shallow weighing bottle was weighed & dried under the same conditions to be employed in the determination; 2 gm of sample was transferred to the bottle. It was covered properly & again weighed. The sample was distributed as evenly as practicable by gentle sidewise shaking to a depth not exceeding 10mm & the loaded bottle was placed in the oven. The stopper was removed and it was also leaved in the chamber. The sample was dried to constant weight. After drying was completed, the bottle with sample was allowed to cool at room temperature in desiccators before weighing. The percentage loss on drying was calculated with reference to the air-dried drug.

EXTRACTION OF PLANT MATERIAL

Selection of Solvent: As per literature review & the nature of phytochemicals present in drug as well as on the basis of their polarity, the solvents were selected for the extraction of the leaves of Barleria prionitis Linn. Like Petroleum-ether (60-800C), ethyl acetate, methanol.

Selection of Extraction method:

According to the literature survey & nature of phytochemicals present in drug, the extraction method was selected. The extraction method selected for extraction from the leaves of Barleria prionitis Linn. Was continuous hot extraction method using soxhlet apparatus. The method was selected for its efficiency. Factors which are considered for selection of extraction method were temperature, time, economy, completion of extraction etc. Out of these temperature is the rate-limiting factor for the extraction method. Constant temperature would increase the efficiency of the extraction by increasing the infusibility of the solvent.

Material used:

Soxhlet apparatus, heating mental, powdered drug, Petroleum-ether (60-800C), ethyl acetate, methanol.

Figure 05: Extraction of Barleria prionitis Linn leaves

Procedure:

Extraction of Barleria prionitis Linn.leaves was carried out by continuous hot extraction method in Soxhlet extractor.

Phytochemical Qualitative analysis

Qualitative chemical tests were carried out for all four extracts to identify the presence of various chemical constituents and presented them in table 02.

Development of TLC fingerprints profile of the extracts:

All the extracts of selected plant material were subjected to TLC studies using various solvent systems to determine the presence of various phytoconstituents. The Rf values of observed compounds were noted for all extracts. The characteristic fingerprint of the various chemical constituents in each extract under UV light and after derivatization with suitable reagents was recorded.  Preliminary phytochemical screening revealed the presence of carbohydrate, proteins flavonoids, alkaloids, fixed oils, steroid & saponins. Compounds of varying polarity in the extracts well separated using various solvent systems on TLC. Rf value of the separated compounds were recorded and given in Table No. 5  and some images of TLC are given in Fig no.7  (The Indian Pharmacopoeia, 2006)

Total Polyphenolic Content

Procedure: 4 ml of Folin Ciocalteu reagent was mixed with 1 ml of extract solution, this solution mixture was kept on standing for 5 min & then 5 ml of sodium carbonate was added to it. The absorbance of reaction mixture was measured against blank (without extract) at 765 nm using UV-Visible spectrophotometer. Gallic acid was used as standard for determination of total polyphenol content of extract. The calibration curve was drawn using various concentrations of gallic acid (50, 100, 150, 200, 250 µg/ml). The total polyphenol content was expressed as gallic acid equivalent in mg/g of the extract & was calculated by using following equation obtained from standard gallic acid graph  ( r2 = 0.9918) (Swamyet al, 2012). Results are given in table.

Absorbance (y) = mx + c

Total Flavonoid Content

Procedure: 1 ml of extract solution was mixed with 4 ml of distilled water & 0.3 ml of NaNo2. After 5 min 0.3 ml of AlCl3& 2 ml of NaOH was added, at last total volume was made up to 10 ml with distilled water. The solution was mixed well & absorbance of the solution mixture was measured at 510 nm against prepared blank (without extract). Rutin was used as standard for determination of total flavonoid content of extracts. The calibration curve was drawn using various concentrations of rutin (100, 200, 300, 400, 500 µg/ml). The total flavonoid content was expressed as rutin equivalent in mg/g of the extract & was calculated by using following equation obtained from standard rutin graph (r2 = 0.9964) (Ekramulhaqueet al, 2011).Results are given in table.

Absorbance (y) = mx + c

OBSERVATIONS & RESULTS

 Pharmacognostic Evaluation

Physicochemical Parameters:

The powdered drug was evaluated for its physicochemical parameters like total ash values, acid insoluble ash, water soluble ash &loss on drying. All the results are tabulated in following table 03.

PHYTOCHEMICAL SCREENING OF PLANT EXTRACTS 

TLC Finger printing

Total Polyphenolic content

Table 06: Calibration Curve of Gallic acid

Figure 08 : Calibration Curve 

Table 07 : Total Polyphenolic content of Barleria prionitis; Linn.leaves extracts