INTRODUCTION:

Human beings have been using plant products as herbal medicines since time immemorial. These phytocomponents of medicinal importance are derivatives of plant parts like leaves, flowers, roots, fruits, barks and seeds. In recent years plant based herbal medicines are more prevalent in prevention of many diseases as the plant based natural drugs have less side effects, and are safe and easily degradable in comparison to synthetic drugs¹.

Adhatoda vasica plant belongs to Acanthaceae family, growing throughout India, specifically in the Himalayan regions. The name, Justicia adhatoda and Adhatoda vasica are used synonymously. It is also known as Vasaka or Malabar nut. It is an erect, terrestrial, perennial shrub.

The leaves of the plant are dark green and yellow below from upper to lower surface and flowers are white in colour. Adhatoda vasica is a valuable medicinal plant, used in the treatment of respiratory diseases like asthma, cough, and bronchitis². The different plant parts are being utilised for the treatment of various diseases like joint pain, sprains, eczema, malaria, swellings, tuberculosis and liver diseases^3,4,5.

Phenolics are the largest group of phytochemicals that account for most of the biological functions in plants or plant products. Flavonoids are the naturally occurring phenolic compounds, which are found both in free state and as glycosides in different plant parts. Phenolic and flavonoid compounds act as iron chelator which have antioxidant and radical scavenging properties⁶. These bioflavonoids have the pharmacological activities such as heart stimulant, inhibition of histamines and blockage of inflammatory action of hepatotoxins. Phenolic and flavonoid compounds have been reported to have multiple biological effects such as anti-inflammatory, anti-carcinogenic, antioxidant and free radicals scavenging abilities⁷. They are found to contain many biological activities including antimicrobial, antiulcer, anticancer, antiarthritic, antiangiogenic, inhibition of protein kinase and inhibition of mitochondrial adhesion.

Medicinal plants contain antioxidants which play important role as health-protecting factor. The antioxidants protect plants from external damage. Antioxidant compounds have an ability to remove free radicals in human body and also prevent the deterioration of fats and other ingredients of food. Antioxidants have the ability to reduce the chances of chronic diseases such as cancer and heart problems. Deficiency of antioxidants in human is associated with variety of health problems, viz. cardiovascular diseases, diabetes, cataracts, rheumatoid arthritis, and Alzheimer’s disease. Most of the antioxidant compounds are derived from plant sources in dietary supplements. These antioxidant compounds are being utilised commercially and thus are being attracted by many researchers due to their economic viability⁸. Various methods have been developed for analysis of antioxidants in medicinal plants. The stable free radical diphenyl picryl hydrazyl (DPPH) is being utilised mostly for analysis of antioxidant in medicinal plants⁹. The basic antioxidants suggested by clinical and epidemiological research include Ascorbic acid (Vitamin C), Anthocyanin, lutein, β-carotene, Lycopene, butylated hydroxy toluene (BHT), and chlorophyll¹⁰.

Gas Chromatography-Mass Spectroscopy (GC-MS) is a very harmonious technique, which is used for the identification and quantification of compounds. The unknown compounds in a complex mixture can be identified by analysis of the data and also by matching the identical mass spectra with reference spectra¹¹.

This study was subjected for the preliminary phytochemical analysis, quantification of the total phenolic content and flavonoid content in Adhatoda vasica plant methanolic extracts, antioxidant activity of plant extract and GC-MS profiling of Adhatoda vasica leaf, stem and flower extracts for the identification and characterization of phytocompounds.

MATERIALS AND METHODS:

Collection and preparation of plant material:

The plant was collected from Kanak Ghati of Jaipur, Rajasthan, India. The plant Adhatoda vasica was identified by Herbarium, Department of Botany, University of Rajasthan, Jaipur. The plant specimen was submitted to Herbarium for accession number. The collected plant was washed in running tap water for removal of dust. After washing, the leaf and flowers were cut into small pieces, separated and shade dried. The dried plant parts were then powdered using a mixer grinder and the plant powder was preserved in vacuumed polythene cover till further use.

Preparation of samples:

The plant leaf, stem and flower powder (100gm) were extracted with 80% methanol using Soxhlet extractor for 24 h. The extracts were filtered using Whatman’s No.1 filter paper. The residue obtained after the filtration was again extracted in ethyl ether and ethyl acetate using a separating funnel. The extracts were separated in ether layer and then ethyl acetate in a separating funnel. Then the final layer was extracted using 7% H₂SO₄ and ethyl acetate. These layers were filtered. The final layers were then concentrated by heating at 60°C on water bath. The extracts thus obtained were then analyzed by Gas Chromatography Mass Spectrometer (GC-MS).

Preliminary phytochemical analysis:

Assessment of the various phytochemicals in Adhatoda vasica leaf, stem and flower extracts was done using standard procedures as described by Harborne, (1973)¹² and Solomon et al. (2013)¹³. The preliminary phytochemical screening of plant extract was carried out to determine the presence of phenols, flavonoids, reducing sugars, saponins, aldehyde and ketones, terpenoids, saponins and cardiac glycosides.

Estimation of total phenolic content:

The total phenolic content was estimated using the Folin Ciocalteu colorimetric method¹⁴. Various concentrations of Gallic acid solutions in methanol (20, 40, 60, 80 and 100 µg/ml) were prepared. An aliquot of 1 ml Gallic acid of each concentration was added to tube containing 5 ml of (10%) Folin-Ciocalteu reagent and 4 ml of 7% Na₂CO₃ were added to get a final volume of 10 ml. The mixture was shaken and kept for90 minutes at room temperature. Then the absorbance was measured at 720 nm by UV Visible spectrophotometer against Gallic acid. Total phenolic content was denoted as mg Gallic Acid Equivalents (GAE).

Estimation of total flavonoid content:

Total flavonoid content was measured by the Aluminium Chloride colorimetric assay¹⁵. In this method Quercetin was used as standard. 1mlof extract or standard solution of quercetin in series of 20, 40, 60, 80 and 100 (µg/ml) was taken in 10 ml volumetric flask containing 4 ml of distilled water. To the flask, 0.3 ml of 5% NaNO₂was added. After five minutes, 2 ml 1M NaOH was added and final volume made up to 10ml by adding distilled water. The absorbance was recorded at 510nm using UV-Visible spectrophotometer.

Free Radical Scavenging Activity Test (DPPH Method)

The antioxidant activity of the plant extracts and the standard was assessed by quantifying the scavenging ability to stable free radical DPPH (1,1-diphenyl-2- picrylhydrazyl)¹⁶.

The plant extract was prepared in methanol as sample solution. Ascorbic acid solution prepared in double distilled water was used as standard solution and 0.002% of DPPH was prepared in methanol. 1 ml of DPPH solution was added to 1 ml of sample and 1 ml of standard solution separately. These solution mixtures were kept at room temperature in dark for 1hour. The disappearance of DPPH was read at 525 nm using spectrophotometer. Methanol was used as blank for plant extract sample and double distilled water was used as a blank for ascorbic acid. The optical density was recorded and % inhibition was calculated using the following formula:

A-B

I= ------------x100

Where A is the OD of the control and B is OD of the sample.

GC-MS analysis of plant extracts:

GC-MS technique was performed using SHIMADZU QP2010 ULTRA system containing Elite-1 fused silica capillary column. Helium gas was employed as carrier gas at a constant flow rate of 1.51ml/min and a volume of 2µ was injected. An electron ionization energy system with ionization energy of 70eV was used. The Injector temperature was 200°C and the oven temperature was programmed from 70°C with an increase of 300°C for 10 min. Mass spectra were taken at 70eV with a scan interval of 0.5 seconds. Total GC running time was 30 min. The amount of each compound was calculated by percentage of peak area of compound out of the total area.

RESULTS:

Preliminary phytochemical analysis:

Preliminary detection of phytochemicals in the plant extracts was done by applying standard phytochemical tests for saponins, phenols, flavonoids, terpenoids, steroids, aldehyde, ketones, reducing sugar, glycosides and resins. The plant showed both presence and absence of various compounds (Table 1). In the preliminary analysis of phytochemicals present in plant extracts of Adhatoda vasica leaf, stem and flower were positive for the most of the phytochemical constituents namely saponins, phenols, flavonoids, terpenoids, steroids, aldehyde and ketones. The leaves were found negative for glycosides and resins; the stems were negative for reducing sugars, glycosides and resins and flowers had absence of reducing sugar, glycosides and saponins.

Table 1: Preliminary phytochemical screening of Adhatoda vasica

Sr. No.	Test	Leaf	Stem	flower
1	Flavonoid test	+ve	+ve	+ve
2	Phenolic test	+ve	+ve	+ve
3	Reducing sugars test	+ve	-ve	-ve
4	Aldehyde & keton test	+ve	+ve	+ve
5	Glycosides test	-ve	-ve	-ve
6	Terpenoids test	+ve	+ve	+ve
7	Saponinstest	+ve	+ve	-ve
8	Resins test	-ve	-ve	+ve

+ve :Positive, -ve : Negative

Analysis of Total phenolic and flavonoid content:

The results for total phenolic and total flavonoid content are presented in Table 2. The results showed that Adhatoda vasica leaf, stem and flower are rich sources of phenolic and flavonoid content. The highest total phenolic content (TPC) was observed for the leaves of Adhatoda vasica (38.1±1.32mg GAE) followed by flowers (31.50±0.91mg GAE) and stems (26.8 ±0.72mg GAE). Whereas in flavonoids, maximum flavonoid content was found in Adhatoda vasica flower extract (42.2 ±0.032 mg QE) followed by leaf (35.9 ±0.14mg QE) and stem (22.20±010mg QE).

Table 2: Total phenolic and flavonoid content in Adhatoda vasica

Plant part	Total Phenolic content (mg/g GAE)	Total flavonoid (mg/g QE)
Leaf	38.1 ± 1.32	35.9 ±0.87
Stem	26.8 ±0.72	22.20 ± 2.10
Flower	31.50± 0.91	42.2 ± 1.14

*GAE- Gallic Acid Equivalent; QE- Quercetin Equivalent

Fig 1: Total Phenolic and flavonoid contents of Adhatoda vasica leaf, stem and flower extract

Antioxidant activity:

The Antioxidant activity of plant extracts of A. vasica was measured by the decrease in the absorbance of a stable free radical DPPH. It was observed that A. vasica flower showed higher antioxidant activity at all concentrations than leaves and stems (Table 3).

Fig 2: DPPH radical scavenging activity of Adhatoda vasica leaf, stem and flower extract

Table 3: DPPH radical scavenging activity of Adhatoda vasica leaf, stem and flower

Conc. (µg/ml)	% inhibition of leaf extract	% inhibition of stem extract	% inhibition of flower extract
20	15	12	18
40	32	23	37
60	49	35	54
80	62	48	69

Identification of plant compounds through GC-MS:

The identification of chemical components from the Adhatoda vasica leaf, stem and flower extracts by GC-MS analysis was conducted using the National Institute Standard and Technique (NIST) database having more than 2,00,000 patterns. The chromatogram of unknown component was compared with the mass spectrum chromatograms of the known components stored in NIST-11 library. The database compared using GC-MS software GC-SOLUTION. The GC-MS Chromatograms of Adhatoda vasica leaf, stem and flower extracts showed the presence of various phytocompounds (Figure 3,4 and 5). The compounds were identified using Similarity Index of compound in GC-MS library. Similarity Index is the similarity of test compound to any compounds stored in GC-MS library. The Molecular weight and Molecular formula were also used for the identification of compounds.

Table 4: Phytocompounds identified in Adhatoda vasica leaf ethyl acetate extract

Peak No.	Retention Time	Area	Area%	Name	Similarity Index	Molecular Weight
1.	9.051	138170	0.73	Pentadecane	96	212
2.	9.112	204959	1.09	3-Hexadecene, (Z)-	96	224
3.	12.400	302830	1.61	Methyl tetradecanoate	93	242
4.	12.431	424420	2.25	1-Heptadecene	96	238
5.	13.835	6416876	34.02	Hexadecanoic acid, methyl ester	96	270
6.	14.501	417925	2.22	Hexadecanoic acid, 15-methyl-, methyl ester	95	284
7.	15.108	1318264	6.99	9-Octadecenoic acid, methyl ester, (E)-	94	296
8.	15.143	1802724	9.56	Methyl stearate	93	298
9.	15.179	3881287	20.58	9,12-Octadecadienoic acid (Z,Z)-, methyl ester	93	294
10.	15.320	1542470	8.18	9,12,15-Octadecatrienoic acid, (Z,Z,Z)-	95	292
11.	16.341	235122	1.25	Methyl 18-methylnonadecanoate	90	326

Fig 3: GC-MS Chromatogram of Adhatoda vasica leaf ethyl acetate extract

Table 5: Phytocompounds identified in Adhatoda vasica stem ethyl acetate extract

Peak No.	Retention Time	Area	Area%	Name of compound	Similarity Index	Molecular Weight
1.	9.115	233663	3.78	3-Hexadecene, (Z)-	96	224
2.	10.823	154762	2.50	Undecanoic acid, 10-methyl-, methyl ester	80	214
3.	10.867	315564	5.10	3-Octadecene, (E)-	97	252
4.	12.369	94325	1.53	1-Bromo-3,7-dimethyloctane	63	220
5.	12.433	286652	4.64	1-Heptadecene	96	238
6.	13.494	146143	2.36	8-Octadecanone	88	268
7.	13.836	1505741	24.35	Hexadecanoic acid, methyl ester	96	270
8.	14.841	186639	3.02	Hexadecanoic acid, 2-hydroxy-, methyl ester	87	286
9.	15.111	695617	11.25	9-Octadecenoic acid, methyl ester, (E)-	96	296
10.	15.145	388358	6.28	Heneicosanoic acid, methyl ester	90	340
11.	15.181	801933	12.97	9,12-Octadecadienoic acid (Z,Z)-, methyl ester	91	294
12.	15.321	274182	4.43	9,12,15-Octadecatrienoic acid, (Z,Z,Z-	94	292
13.	17.977	170870	2.76	Benzenesulphonic acid, 4-(5-decyl)-,	73	312

Fig. 4: GC-MS Chromatogram of Adhatoda vasica stem ethyl acetate extract

Table 6: Phytocompounds identified in Adhatoda vasica flower ethyl acetate extract

Peak No.	Retention Time	Area	Area%	Name	Similarity Index	Molecular Weight
1.	6.430	332866	9.20	Pentanoic acid, 4-oxo-, methyl ester	96	130
2.	9.114	482876	13.53	3-Octadecene, (E)-	96	252
3.	11.670	99036	2.74	Nonanedioic acid, dimethyl ester	80	216
4.	12.368	189573	5.24	Urs-12-en-28-ol	58	426
5.	12.431	245758	6.80	1-Heptadecene	96	238
6.	13.494	104998	2.90	8-Pentadecanone	88	226
7.	13.834	325925	9.01	Hexadecanoic acid, methyl ester	92	270
8.	15.144	165074	4.56	Heneicosanoic acid, methyl ester	88	340
9.	17.299	95009	2.63	Benzenesulphonic acid, 4-(5-undecyl)-	81	326
10.	17.439	164595	4.55	Benzenesulphonic acid, 4-(4-dodecyl)-	75	340
11.	17.901	107596	2.98	Benzenesulphonic acid, 4-(6-dodecyl)-	76	340
12.	17.972	104538	2.89	Benzenesulphonic acid, 4-(5-decyl)-	75	312
13.	18.176	263822	7.29	Cycloheptanone, 2-(phenylmethylene)-	63	200
14.	18.492	161200	4.46	Benzenesulphonic acid, 4-(3-dodecyl)-	93	340
15.	19.056	172244	4.76	6-Bromotricyclo[8.2.2.2(4,7)]hexadeca-1(13),4,6,10(14),11,15-hexaen-5-ol	66	302

Fig. 5: GC-MS Chromatogram of Adhatoda vasica flower ethyl acetate extract

The GC-MS analysis of flower extract revealed the existence of 19 compounds (Table 6). In the total ion chromatogram (TIC), the major compounds were 3-Octadecene, (E)- (13.53%), Pentanoic acid, 4-oxo- (9.20%), Hexadecanoic acid (9.01%), Cycloheptanone, 2-(phenylmethylene)- (7.29%), 1-Heptadecene (6.80%), Urs-12-en-28-ol (5.24%), 6-Bromotricyclo [8.2.2.2(4,7)] hexadeca-1(13),4,6,10(14),11,15-hexaen-5-ol (4.76%), Heneicosanoic acid (4.56%), Benzenesulphonic acid, 4-(4-dodecyl)- (4.55%), Benzenesulphonic acid, 4-(3-dodecyl)- (4.46%), Benzenesulphonic acid, 4-(6-dodecyl)- (2.98%) and Benzenesulphonic acid, 4-(5-decyl)- (2.89%).

DISCUSSION:

The plant A. vasica leaf, stem and flower extracts were analyzed for phytochemical analysis and revealed the presence of most of the medicinal important compounds viz phenolic, flavonoids, terpenoids, saponins and aldehydes by preliminary analysis and GC-MS screening. The plant Adhatoda vasica has great potential for curing asthma, bronchitis, and cough and rheumatism due to the presence of various phyto-compounds¹⁷. Similarly, Kumar et al., (2014) studied the presence of chemical compounds tannins, alkaloids, saponins and flavonoids in A. vasica leaf extract and established their role in treatment of dysentery and diarrhea¹⁸.

The phenolic and flavonoid compounds are plant polyphenols which are the most widely distributed secondary metabolites in plant kingdom in high concentrations. The plant polyphenols, secondary metabolites, are widely distributed in the plant kingdom and are sometimes present in surprisingly high concentrations¹⁹. The phenolic compounds neutralize free radicals by donating a hydrogen atom or electron²⁰. The results of present study showed that the amount of phenolic contents varied in different parts of plants. The leaves and flowers showed significant quantity of phenolic and flavonoid compounds whereas stems exhibited comparatively lower quantity of phenolic and flavonoid content.

The plant Adhatoda vasica leaf, stem and flower methanol extracts were evaluated by scavenging capability of DPPH and measured using spectrophotometer. After scavenging of DPPH by Ascorbic acid, the DPPH violet colour gets disappear. This colour change was used for the analysis of antioxidant activity of plant extracts²¹. In our study, the plant leaves, stems and flowers exhibited the different scavenging capacity at varied concentrations. The maximum antioxidant activity of methanolic extract was observed at 80μg/ml and minimum at 20μg/ml²². However, the highest antioxidant activity was observed in flower extract at all concentration indicating that it contains more phenolic and flavonoid content as well²³. Antioxidant activity has been assayed in a number of Adhatoda species including Adhatoda vasica and Adhatoda beddomei. The extracts from Adhatoda beddomei showed 80% inhibition of free radicals at 100μg/ml concentration²⁴. It has been reported that the antioxidant activity of many plants depend on their phenolic content. The antioxidant activity of flower in this study may be due to the presence of high content of phenolic and flavonoid compounds in flowers²⁵.

In the phytochemical screening of Adhatoda vasica leaf, stem and flower extracts, through GC-MS, the plant parts showed the existence of many phytocompounds. These phytocompounds present in plant belong to fatty acids or terpenoids. These bioactive compounds present in plant leaf, stem and flowers are used for the treatment of various infections and diseases by traditional practitioners²⁶.

The compounds identified from Adhatoda vasica in the present study are of medicinal importance. Octadecanoic acid have been reported to exhibit antibacterial and antifungal activity and also used for the treatment of tuberculosis, diabetic mellitus, respiratory tract infections and vaginal infections^27,28. Different researchers revealed the use of n-hexadecanoic acid as antioxidant, anti-cancer, anti-microbial, anti-haemolytic, anti-diabetic compound²⁹. Similarly, tetradecanoic acid compound also exhibited antibacterial and antioxidant properties. Furthermore, tetradecanoic acid inhibits cell proliferation and exhibits nematocidal activity^30,31.

CONCLUSIONS:

Adhatoda vasica is an important medicinal plant used for the treatment of whooping cough, asthma and bronchitis. The plant and its parts were screened for the presence of important phyto-compounds by preliminary phytochemical analysis using standard protocol. The scientific exploration of the plant included quantification of total phenolic and flavonoid content along with free radical scavenging activity and showed the existence of some important phytochemical constituents in the plant extracts. These plant part extracts were further subjected to GC-MS analysis. In GC-MS analysis of Adhatoda vasica leaf, stem and flower extract, many phyto-compounds were reported. The major compounds reported in Adhatoda vasica, plant extracts include Hexadecanoic acid, Octadecatrienoic acid, Heneicosanoic acid, Benzenesulphonic acid, Cycloheptanone and 1-Heptadecene. Thus, from the results obtained it can be concluded that Adhatoda vasica is a therapeutically important plant and can be suggested for treatment of many infections and diseases.

CONFLICT OF INTERESTS:

We declare that there is no conflict of interest.

ACKNOWLEDGMENT:

The authors extend their sincere thanks to the authorities of JECRC University, Jaipur, India for providing necessary research facilities and financial support for completion of this work.

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Received on 04.05.2019 Modified on 10.06.2019

Research J. Pharm. and Tech. 2019; 12(12): 5699-5705.

DOI: 10.5958/0974-360X.2019.00986.7