INTRODUCTION:

Thousands of years ago, natural products, especially herbs, are used for the treatment of various diseases. Nowadays, plants are staging a comeback, and herbal renaissance is happening all over the globe. According to WHO, 75-80% of the world population, mainly in developing countries, depend on medicinal herbs for primary health care (Larkin, 1983). This is primarily because of the general belief that herbal remedies are without any side effects beyond being cheap and locally available. On the report of the World Health Organization (WHO), the use of herbal remedies throughout the world be more than that of the conventional drugs by two to three times [1].

Exacum bicolor Roxb. is an angiosperm belonging to the cosmopolitan family Gentianaceae, which is a family of flowering plants with 84 genera and 1688 species. The plants of this family are perennial herbs or shrubs. Exacum bicolor Roxb. is an erect herb growing from July to November in plains and July to January in high ranges. The flowering period of this plant in plain from September to November, whereas in high altitude, flowering will bear up to January. The unique feature of this plant is limited to a small area only even in a vast stretch of grasslands, and the frequency of distribution is more in the dry pastures of plain compared to high altitude grasslands. In the plain dry grassland, it grows up to a height of 25-80cm. In high altitude grassland, it grows up to 40-120cm. The whole plant having benefits such as, as a tonic, febrifuge, and stomachic. The plant also yields a dye. Being bitter in taste, the local people take it as an herbal remedy for diabetes and skin disorder. In Kerala, the traditional practitioners prescribe decoction of the whole plant for washing the eyes. However, due to the limited distribution and lack of availability of the plant through out the year, it is not commonly used [2].

Pharmacognostic study ensures plants identity, lays down standardization parameters, which will help and prevent adulteration. Such reviews will aid in the authentication of the plants and ensure reproducible quality of natural products which will lead to safety and efficacy of natural product [3].

MATERIALS AND METHOD:

Plant authentication and extraction process:

The whole plant of Exacum bicolor Roxb. (Family: Gentianaceae) were collected in the month of August-November from Kasargod and Kannur district, Kerala, India. The plant was taxonomically identified and authenticated. A voucher specimen (17PH001R) was placed for future reference in Pharmacognosy laboratory of Department of Pharmacognosy, Nitte Gulabi Shetty Institute of Pharmaceutical Sciences, Karnataka, India. The whole fresh plant was collected, washed and shade dried for one week at a temperature not exceeding 60⁰C to prevent the deactivation of thermolabile phytoconstituents. The dried plant was coarsely powdered, sieve through 60mesh and stored in an airtight container at room temperature until further use. The powdered Exacum bicolor was used for powder analysis and physicochemical parameters. For phytochemical screening, the powdered drug (200gm) was extracted successfully by using petroleum ether, chloroform, ethyl acetate, methanol, and water by maceration. All the extracts were concentrated using vacuum rotary evaporator and dried in a vacuum desiccator.

Pharmacolognostical Studies:

Physico-chemical parameters:

Physico-chemical parameters of dried whole plant powder, such as moisture content, extractive value, ash value, foaming index, swelling index, foreign matter, fluorescence analysis using various reagents were performed as per the official standard procedure is given in Ayurvedic Pharmacopoeia 1999.

Phytochemical Evaluation:

Preliminary phytochemical screening of all extracts was performed to evaluate the presence of various phytochemicals like alkaloids, glycosides, flavonoids, steroids/triterpenoids, phenolics, tannins, saponins, mucilages, protein, amino acids and carbohydrates as per the standard procedure.

Quantitative Estimation:

a) Total Phenolic content:

The total phenolic content of the various extracts was determined by the Folin Ciocalteu method. 200μl of crude extracts (1mg/ml) was made up to 3ml with distilled water, mixed thoroughly with 500μl of Folin Ciocalteu reagent for 3min, followed by the addition of 2ml of 20% w/v sodium carbonate. The mixture was allowed to stand for further 60min in dark and absorbance was measured at 650nm. The calibration curve of gallic acid was used to calculate the total phenolic content, and the results were expressed as mg of gallic acid equivalent per gram dry weight [4].

b) Total Flavonoid content:

The total flavonoid content of the Exacum bicolor extract was determined by the aluminum chloride colorimetric method. 50μl of crude extract (1mg/ml) was made up to 1ml with methanol, mixed with 4mL of distilled water and then 0.3ml of 5% sodium nitrite solution; 0.3mL of 10% aluminum chloride solution was added after 5min of incubation, and the mixture was allowed to stand for 6min. Then, 2ml of 1mol/l sodium hydroxide solution was added, and the final volume of the mixture was brought to 10ml with double-distilled water. The mixture was allowed to stand for15min, and absorbance was measured at 510nm. The total flavonoid content was calculated from a calibration curve, and the result was expressed as mg rutin equivalent per g dry weight [5].

c) Total Alkaloidal content:

The plant extract was dissolved in DMSO, added 1ml of 2N hydrochloric acid and filtered. This solution was transferred to a separating funnel, 5ml of bromocresol green solution, and 5ml of phosphate buffer (p^H 4.7) were added. The mixture was shaken with 1, 2, 3 and 4ml chloroform by vigorous shaking and collect in a 10ml volumetric flask and diluted to the volume with chloroform.

A set of reference standard solution of atropine was prepared in the same manner as described earlier. The absorbance for test and standard solutions were determined against the reagent blank at 470nm with an UV/Visible spectrophotometer. The total alkaloidal content was expressed as mg of AE/g of extract. All experiments were performed thrice; the results were averaged and reported in the form of mean ± SD [6].

D) Total Saponin content:

20gm of plant sample was dispersed in 200ml of 20% ethanol. The suspension was heated over a hot water bath for 4h with continuous stirring at about 55⁰C. The mixer was filtered and residue re-extracted with another 200ml of 20% ethanol. The combined extracts were reduced to 40ml over water bath at about 90⁰C. The concentrate was transferred into a 250ml separating funnel and 20ml of diethyl ether was added and shaken vigorously. Collect the aqueous layer and repeated the purification twice. Sixty ml of n-butanol was added and the butanol extract was washed twice with 10ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation the sample were dried in the oven to a constant weight. The saponin content was calculated in percentage [7].

RESULTS:

Extraction:

The whole plant material was extracted by using petroleum ether, chloroform, ethyl acetate, methanol and water. The colour, consistency and percentage yield were given in the table no:1.

Physico-chemical parameters:

The pharmacognostic studies such as loss on drying, alcohol-soluble extractive, ether soluble extractive, water-soluble extractive, total ash, water soluble ash, insoluble acid ash, foreign matter, swelling index, foaming index, bulk density, crude fiber contents were determined and the results were given in table no:2. The results of fluorescence analysis was given in table no:3.

Phytochemical evaluation:

The phytochemical screening of all the extracts was carried out, and it showed the presence of alkaloids, flavonoids, phenolic compounds, glycosides, carbohydrates, terpenoids, and saponins. The results were given in the table no:4.

Quantitative estimation:

The total phenolic content, the total flavonoid content of various extracts were determined by Folin Ciocalteu method and Aluminium chloride colorimetric assay method, respectively. The total phenolic content of methanol extract was found to be 18.49 mg of GAE/g, followed by aqueous extract 14.57mg of GAE/g, ethyl acetate extract 12.56 mg of GAE/g, chloroform extract 11.49mg of GAE/g and petroleum ether extract 7.79 mg of GAE/g. The calibration curve of gallic acid was given in the fig no:1

The total flavonoid content of methanol extract was found to be 21.06 mg of QE/g, followed by petroleum ether extract 17.61mg of QE/g, chloroform extract 17.25 mg of QE/g, ethyl acetate extract 15.97mg of QE/g and aqueous extract 15.05mg of QE/g. The calibration curve of quercetin was given in the Fig no:2.

The total alkaloidal content of chloroform extract, ethyl acetate, methanol, and water was found to be 55.98mg of AE/g, 37.78mg of AE/g, 15.98mg of AE/g and 20.41mg of AE/g respectively. The calibration curve of atropine was given in the fig no: 5, and the results were given in table No: 3.

Table No:1 Colour, consistency and percentage yield of various extracts of Exacum. bicolor

Sl. No.	The solvent used for extraction	Colour	Consistency	Percentage yield (%w/w)
1.	Petroleum ether	Green	Semi-solid	0.32
2	Chloroform	Dark green	Solid	1.07
3	Ethyl acetate	Dark green	Solid	1.98
5	Methanol	Yellowish-brown	Solid	6.19
6	Water	Brown	Solid	11.34

Table no: 2 Physico-chemical parameters of Exacum bicolor

Sl. No:	Quantitative standards		Observation
1	Moisture content^*		11.55±0.1763%w/w
2.	Extractive value	Ether soluble extractive^*	2.67±0.267w/v
		Alcohol soluble extractive^*	21.330.706w/v
		Water soluble extractive^*	23.46±.267w/v
3	Ash value	Total ash^*	7.33±0.4410w/w
		Acid insoluble ash^*	0.67±0.167w/w
		Water soluble ash^*	3.67±0.167w/w
4	Foaming index		<100
5	Swelling index		33.33%v/v
6	Foreign matter		<0.5%w/w

Each column represents as means ± SD (n = 3)

Table No:3 Fluorescence analysis of Exacum bicolor

Sl.no.	Treatment	Observation under ordinary light	Observation under UV light
1	Powder as such	Light brown	Brown
2	Powder+ 1N NaOH	Yellowish-brown	Green
3	Powder+ acetic acid	Brown	Yellow
4	Powder+ 1N HCl	Light brown	Yellow
5	Powder+ conc. HNO₃	Reddish-brown	Green
6	Powder+ 5% FeCl₃	Bluish green	Bluish green
7	Powder+ 5% Iodine	Dark brown	Bluish-green
8	Powder+ 50% HNO₃	Yellow	Yellow
9	Powder+ 50% H₂SO₄	Brown	Brown
10	Powder+ conc. H₂SO₄	Brown	Brown

Table no:4 Phytochemical analysis of various extracts of Exacum bicolor

Sl No	Phytoconstituents	Pet. Ether extract	Chloroform extract	Ethyl acetate extract	Methanol extract	Aqueous extracts
1.	Alkaloids	-	++	+	++	+
2.	Glycosides	-	-	++	+	+
3.	Phenolics	+	+	++	++	+
4.	Flavones and Flavonoids	-	+	++	++	+
5.	Carbohydrates	-	-	++	+	+
6.	Proteins and Aminoacids	-	-	-	-	-
7.	Terpenoids	+	-	-	-	-
8.	Sterols	+	-	+	+	-
9.	Saponins	-	-	+	+	++
10.	Gums and mucilage	-	-	-	-	+

Table No: 5 Results showing quantitative analysis of Exacum bicolor

Sl. No:	Extracts	Total phenolic content mg of GAE/gm	Total flavonoid content mg of QE/gm	Total alkaloidal content AE/gm	Total saponin %W/W
1	Petroleum ether extract	7.79±0.7300	17.61±0.0762	-	1.1
2	Chloroform extract	11.49±0.2876	17.25±0.7595	55.98±2.8595
3	Ethyl acetate extract	12.56±0.2867	15.97±0.6808	37.78±2.1000
4	Methanol extract	18.49±0.3282	21.06±0.2569	15.98±1.2700
5	Aqueous extract	14.57±0.5682	15.05±0.7269	20.41±1.3163

Each column represents as means ± SD (n = 3)

DISCUSSION:

Standardization of herbal drugs plays an important role to confirm the safety and efficacy of herbal drugs. Herbal drug standardization should be performed by checking various parameters. The amount of active constituents present in the same herbal drugs varies according to various intrinsic and extrinsic factors.

Herbal drug standardization is vital for the safety and efficacy of the drug. Herbal drug standardization should be done through multiple modes as the concentration of the phytochemical varies according to climate, soil, and environment [8]. The moisture contentof the whole plant powder is less, which helps to prevent the microbial attack and deterioration of plant active constituents. The extractive value of crude drugs helps to determine the quality and purity of the drug and for the determination of exhausted or adulterated drugs. Ash contains the inorganic radicals like phosphates, carbonate, and silicates of sodium, potassium, magnesium, calcium, etc. these are present in a definite amount in particular crude drug and these are used to determine foreign inorganic matter present as an impurity [9]. The swelling index of plant indicates the presence of gums and mucilage, pectin and hemicellulose present in the crude drug. The fluorescent analysis of powdered drug also plays an important role in the determination of quality and purity of the drug [10].

The phytochemical screening of various extracts showed the presence of alkaloids, glycosides, phenolic compounds, flavonoids, sterols, carbohydrates, terpenoids, saponins and gums, and mucilage. The methanolic extracts of Exacum bicolor showed the highest amount of total phenol and total flavonoid, which is an important parameter for their antioxidant activity. The chloroform extract contains a significant amount of alkaloid compared to other extracts, and isolation of chloroform extract will help to recognize the nature of alkaloid.

CONCLUSION:

The physicochemical parameters, morphology, microscopy, and phytochemical screening of Exacum bicolor were carried out, and these results serve as a tool for the identification and confirmation of identity and purity of the plant. The determination of total phenolic and flavonoid content showed this plant having a potent free radical scavenging effect. Thus, there is a need for further research in this plant to confirm the pharmacological uses.

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Received on 19.07.2019 Modified on 19.11.2019

Research J. Pharm. and Tech. 2020; 13(4):1752-1756.

DOI: 10.5958/0974-360X.2020.00316.9