Comparative Study on Pharmacognostical, Phytochemical Investigations and Quantification of Vasicine Content in the Extracts of Adhatoda vasica Nees and Adhatoda beddomei CB Clarke

Herbal medicine is the oldest form of healing and treating various diseases.1 Herbal medicines have been widely used due to its more cost effective with no side effects when compared to synthetic drugs. About 80% of the world population rely mainly on herbal medicine for their primary health care.2 The increasing demand in the use of herbal medicine gain an attention of the researchers interestingly to study the standardization and quality control of herbal drugs in order to avoid the erroneous medication and improve the quality of drugs. Standardization is a significant tool for ensuring the quality of herbal drugs. The drug identity, purity, content, chemical and other biological properties determine the quality of the drug.3


INTRODUCTION
Herbal medicine is the oldest form of healing and treating various diseases. 1 Herbal medicines have been widely used due to its more cost effective with no side effects when compared to synthetic drugs. About 80% of the world population rely mainly on herbal medicine for their primary health care. 2 The increasing demand in the use of herbal medicine gain an attention of the researchers interestingly to study the standardization and quality control of herbal drugs in order to avoid the erroneous medication and improve the quality of drugs. Standardization is a significant tool for ensuring the quality of herbal drugs. The drug identity, purity, content, chemical and other biological properties determine the quality of the drug. 3 Tropical species of the genus Adhatoda (Acanthaceae) commonly known as 'Vasa' in India have long been used for the treatment of cold, cough, asthma, bronchitis and tuberculosis. AdhatodavasicaNeesis a sub herbaceous widespread throughout the temperate regions of South Asia, 4 ingredients for the prevention and treatment of recently emerged respiratory infection, Covid-19. Earlier, these medications were used for the treatment of swine flu and dengue fever, respectively. 16,17 Besides, Adhatoda beddomei also have been used for asthma by tribal in Kerala.
GC-MS fingerprint profiling of ethanolic extract of A. beddomei was reported and found to possess various volatile constituents 8 and identification and quantification of alkaloids and flavonoids were also reported on these plants. 18,19 Although, much phytochemical information about these plants were reported, there has been no evidence on pharmacognostical and comparative investigations with the plant A. beddomei, which can be a better alternative species for A. vasica. However, both the plants are closely related to each other and have similar morphological characteristics. The objectives of the present study were to evaluate the comparative pharmacognostical and phytochemical parameters of Adhatoda vasica and Adhatoda beddomei to elucidate the major differences between the two species of Adhatoda. Besides, an attempt has also been made to find out the presence or absence phytoconstituents and quantitative estimation of the pyrroquinazoline alkaloid, vasicine in both the species.

Collection and authentication of plant
The leaves of Adhatoda vasica and Adhatoda beddomei were collected from Chengalpattu and Gingee Hills, Villupuram (Dt), respectively in the month of October 2017. The plants were authenticated by Prof. P. Jayaraman (Botanist), Plant Anatomy Research Centre (PARC), Tambaram, Chennai, Tamil Nadu, India with a reference number of A. vasica and A. beddomei was PARC/2018/3653 and PARC/2018/3654 respectively. The plant specimens were preserved in the Herbarium at Interdisciplinary Institute of Indian System of Medicine (IIISM), SRM Institute of Science and Technology, Kattankulathur, Chengalpattu, Tamil Nadu, India. Collected leaves were cleaned with a running tap water, dried under shade and powdered using cutter mill, sieved through 60 mesh sieve and stored in an airtight container at room temperature.

Chemicals and reagents
Analytical grade hexane, chloroform, ethyl acetate and methanol used for extraction were purchased from Ranchem Private Limited. Formic acid (analytical grade), methanol (HPLC grade) were procured from Ranchem Private Limited. Chemicals of analytical grade and other reagents were obtained from Merck Specialities Private Limited, Mumbai.

Macroscopic analysis
Sensory and macroscopic analysis of both the leaves of A. vasica and A. beddomei were performed and the parameters evaluated for the fresh leaves were shape, size, colour, venation, apex, margin, base, texture, fracture,odour, taste, and petiole size.

Collection of Specimens
The required samples of different organs were cut and removed from the plant and fixed in FAA (formaldehyde: alcohol: acetic acid, 10: 50: 5% with 35% water). After 24h of fixing, the specimens were dehydrated with graded series of tert-butyl alcohol as per the schedule given by Sass, 1940. 20 Infiltration of the specimens was carried by gradual addition of paraffin wax (melting point 58-60°C) until TBA solution attained super saturation. The specimens were cast into paraffin blocks.

Sectioning
The embedded paraffin wax was sectioned with the help of Rotary Microtome. The thickness of the section was 10-12µm. Dewaxing of the section was done by customary procedure. 21 The sections were stained with Toluidine blue as per the method published by O'Brien et al1964. 22 Since Toluidine blue is a polychromatic stain. The staining results were remarkably good; and some cytochemical reactions were also obtained. The dye rendered pink colour to the cellulose walls, blue to the lignified cells, dark green to suberin, violet to the mucilage, blue to the protein bodies etc. Wherever necessary sections were also stained with safranin and fast-green and iodine potassium iodide (for Starch).
For studying the stomatal morphology, venation pattern and trichome distribution, paradermal sections (sections taken parallel to the surface of leaf) as well as clearing of leaf with 5% sodium hydroxide or epidermal peeling by partial maceration employing Jeffrey's maceration fluid 20 were prepared. Glycerin mounted temporary preparations were made for macerated/cleared materials. Powdered materials of different parts were cleared with NaOH and mounted in glycerin after staining. Different cell components were studied and measured.

Photomicrographs
Microscopic descriptions of tissues were supplemented with micrographs wherever necessary. Photographs of different magnifications were taken with Nikon Lab photo 2 microscopic Unit. For normal observations bright field was used. For the study of crystal, starch grains and lignified cells, polarized light was employed. Since these structures have birefringent property under polarized light, they appear bright against dark background. Magnifications of the figures are indicated by the scale-bars.

Physicochemical analysis
Physicochemical analysis such as the percentage of total ash, acidinsoluble ash, water-soluble ash, water and alcohol soluble extractives and loss on drying were determined according to WHO guidelines. 23

Preparation of extracts
Each 5g of powdered A. vasica and A. beddomei were extracted in 50ml of hexane, chloroform, ethyl acetate and methanol for 24h at room temperature for non-successive extraction. The extracts were filtered and re-extracted thrice by following the same procedure. The filtrates were combined and concentrated under reduced pressure using a Buchi rotary evaporator.

Determination of total alkaloid content (TAC)
The extracts were prepared with 1mg/mL of stock solution using 2N hydrocholic acid. 1mL of the solution from the stock was kept in a separating funnel and 1mL of phosphate buffer of pH 4.7 was added for the complex formation for 10min. The complex mixture was partitioned with 3mL of chloroform and the chloroform layer was separated and diluted to 10mL with chloroform. The absorbance of the mixture was measured at 415nm using UV spectrophotometer. Results were expressed as mg of atropine equivalent in g of dry weight of the extract. 27,28 Determination of total phenolic content (TPC) The extract of 100µL solution were mixed with 3.5ml of distilled water and 1mL of Folin-Ciocalteu reagent and kept aside for 8min. To this, 7.5% of 5mL sodium carbonate was added and kept at room temperature for 30min and the absorbance was measured at 765nm. Gallic acid was 2009, using diosgenin as a standard solution. 34 1mL of 80% aqueous methanol was added to 1mL of diluted extracts followed by 1mL of Vanillin reagent and 1mL of 72% sulphuric acid were added to the sides of the tubes. The mixture was heated on a water bath 60°C for 10min and the absorbance was recorded at 544nm against 80% methanol as blank. The saponin content was calculated using standard calibration curve of diosgenin with a concentration range of 20-200µg/mL solution and the results were expressed in mg of diosgenin equivalent to g of dry weight of the extract.

Sample preparation
The extracts of leaves of A. vasica and A. beddomei (each 100mg) were dissolved in 1mL of methanol and sonicated for 15min. The extracts were filtered and made upto 1mL with methanol and stored in a refrigerator until further use.

HPTLC Fingerprinting Analysis
High performance thin layer chromatography (HPTLC) method was performed on pre-coated HPTLC aluminium sheets silica gel 60F 254 plate (10.0 X10.0 cm) of 250µm thickness (Merck, Darmstadt, Germany). The plates were pre-washed with methanol and then activated at 100°C for 15min before use. The prepared sample solutions of 2µL were spotted as 6.0mm narrow bands using 100µL syringe at a constant dosing speed of 150nL/s with a Linomat V semi-automatic applicator (Camag, Muttenz, Switzerland). The HPTLC plate was developed at a distance of 70mm in a 10.0 X10.0 cm twin trough chamber using a pre saturated mobile phase of hexane: ethyl acetate: formic acid (6:4:0.1 v/v/v). The developed TLC plate was dried using hot air for a minute and visualized under 254nm, 366nm and white light using TLC Visualizer. Further, the plate was scanned at the wavelength of 366nm using a TLC Scanner 180712 (Camag) with winCATS Planar Chromatography Manager software.

Quantification of Vasicine by RP-HPLC-PDA
RP-HPLC-PDA method was developed for the quantification of vasicine using Shimadzu LC-20AD HPLC system equipped with CT0-20A controller and the column oven. A Rheodyne 7725 injection valve with 20 µL loop volume, an SPD-M20A photo-diode array detector and a Lab solution 7.1 version software was used for data acquisition and interpretation. Chromatographic separation was achieved on Agilent C 18 column (150mm x 4.6mm; 3µm pore size). The solvent mixture of 0.1%v/v trifluoroacetic acid (A) and methanol: acetonitrile (45:40) (B) with a ratio of (70:30%v/v) at a flow rate 0.6mL/min, the column was maintained at room temperature and the PDA detector was fixed at 280nm.

Macroscopy
Adhatoda vasica and Adhatoda beddomei are sub-herbaceous shrub of family Acanthaceae. Adhatoda vasica, an evergreen, gregarious perennial shrub, 1.2 to 6m in height, distributed throughout India, used as standard and the samples were analysed in triplicate. Total phenolic content was expressed as mg of gallic acid equivalent in g of dry weight of the extract. 29

Determination of total flavonoid content (TFC)
Total flavonoid content was carried out using Aluminium chloride colorimetric assay with slight modifications. 30 The standard calibration curve was plotted with catechin solution (20-100µg/mL). The extract of 100uL was mixed with 4ml of distilled water and 0.3mL of 5% sodium nitrite. After 5min, 0.3mL 10% aluminium chloride solution was added and incubated for 5min at room temperature. Finally, 2mL of 0.1M sodium hydroxide was added and made upto 10mL with distilled water. The formation of pink colour indicated that the formation of complex which further absorbed at 510nm using UV spectrophotometer against blank.

Determination of total terpenoid content (TTC)
Determination of terpenoid content was performed based on the method Ni et al 2012, with ursolic acid as a standard. 31 Calibration curve was constructed using standard concentration of 20-20µg/mL. Initially, 1.5mL of diluted extract was evaporated to dryness at 100°C in a water bath. To the dried residue, 2.5ml of vanillin in acetic acid and 4 mL of glacial acetic acid were added and cooled the solution at room temperature for 15min. The final mixture was absorbed at 548nm against a blank solution using UV spectrophotometer. The terpenoid content was expressed in mg of ursolic acid equivalent in g of dry weight of the extract.

Determination of total glycoside content (TGC)
The estimation of glycoside content was carried out using a Baljet reagent according to the method described in Solich et al 1992. 32 1mL of extract was added to the solution of Baljet reagent containing picric acid and 0.1N sodium hydroxide with the ratio of 95:5. The solution was allowed to incubate in a dark chamber for 60min and the solution was further diluted to 15mL with distilled water and absorbed at 495nm. Digitoxin was used as a standard for the determination of glycoside and the results were expressed in mg of digitoxin equivalent to g of dry weight of the extract.

Determination of total steroidal content (TSC)
The determination of steroidal content was carried out according to Liebermann-Burchard colorimetric assay method 33 with minor modification using cholesterol as a standard. The Liebermann-Burchard reagent was prepared by adding 5ml of concentrated sulphuric acid to 50mL of acetic anhydride solution. The extracts were diluted with chloroform and to the chloroform extract, freshly prepared Liebermann-Burchard reagent was added and measured at 650nm against a reagent blank. Steroidal content was expressed in mg of cholesterol equivalent to g of dry weight of the extract.

Determination of total saponin content (TSAC)
The total saponin content was determined based on Xu and Chang up to an altitude of 1300m. Leaves were elliptical lanceolate or ovate lanceolate in shape, entire, crenate margin and symmetric base, 5-30cm long, hairy, light green in colour at the upper surface and dark green in colour at the lower surface with characteristic odour. A. beddomei, a glaborous shrub of 1 to 3m in height rarely found in higher altitudes of Western Ghats of Kerala and Tamil Nadu. The leaf was simple, entire, wavy, ovate lanceolate in shape, attenuate at base and acuminate apex, 6 to 14cm long and 3 to 4.5mm broad with prominent midrib at the lower surface. Odour was not characteristic and slightly bitter in taste.
Macroscopic characters were studied, compiled and presented in Table  2.

Transverse section of midrib-entire view
In transectional view, the midrib of A. vasica showed bowl-shaped and thick in abaxial part with short thick two adaxial humps and a shallow median concavity (Figure 1a). In A. beddomei, the leaf appeared dorsiventral with thick midrib and bilateral lamina. The midrib was plano-convex in sectional view with semicircular abaxial part and slightly raised adaxial part with slightly wide and shallow adaxial cavity

Lamina
The lamina of A. vasica was dorsiventral with distinction into adaxial and abaxial sides. The lamina was smooth and even on both surfaces of A. beddomei. The lamina was 270µm thick. Adaxial epidermal cells were larger, cubical and thin walled. The abaxial epidermal cells were smaller and rectangular in shape in A. vasica whereas the adaxial epidermal cells and abaxial epidermal cells were circular or squarish and thin walled in A. beddomei. The adaxial epidermal cells contained a prominent cuticle in A. beddomei.    The mesophyll tissue of A vasica was differentiated into adaxial band of thick rectangular palisade mesophyll cell and abaxial zone of 4 or 5 layers of large spherical spongy mesophyll cells. The mesophyll tissue consisted of an abaxial band of 2 short, cylindrical palisade mesophylls were seen in A. beddomei. Small air chambers were present in the spongy mesophyll zone of A. vasica (Figure 4a) and the spongy mesophyll was circular, intercellular air space with stomata occurred on the abaxial epidermal layer of A. beddomei (Figure 4b).

Epidermal trichomes
In A. vasica, two types of epidermal trichomes were arised from epidermal cells. Some are non-glandular type which are multicellular, uniseriate and unbranched. The cells of the trichome were vertically elongated and thin walled. These trichomes were 150µm long and 15µm thick ( Figure 5a) and glandular trichomes have short stalk cell with which it was attached on the epidermis (Figure 5b). The upper secretary body was two celled with dense accumulation of cell inclusions. The glandular trichomes was 10 µm broad and 15µm in height.
In A. beddomei, short prominent glandular trichomes were seen on the abaxial epidermal layer. The trichome was buried in the shallow epidermal cavity which were multicellular and funnel shaped. The cells were darkly stained and the gland was at the level of epidermal layer (Figure 5c).

Crystal distribution
In the mesophyll tissues of A. beddomei, calcium oxalate crystals were seen which was absence in A. vasica. The crystals may be raphide type consisting of several thin needles (Figure 6a) or it could be rectangular long crystals were aggregated in the form of circular cluster (Figure 6b).

Powder microscopy
Trichomes Adhatoda vasica were observed with more non glandular covering type of trichomes on the leaf, stem and petiole surfaces and less frequent glandular trichomes. The non-glandular trichomes were long,  multicellular, unbranched with thick walls and tapering ends ( Figure  7a). The cells of the trichomes are vertically elongated with wide lumen. The trichomes were 130µm long and 10µm thick (Figures 7a and 7c). The glandular trichomes were large, spherical, multicellular (usually 4 celled) secretory head with dense cytoplasm. The gland was subsessile with very short, non-glandular stalk. The glandular head may be 80µm in diameter and capitate type (Figure 7d).
In A. beddomei,short prominent glandular trichomes were seen on the abaxial epidermal layer with multicellular and funnel shaped. The cells were darkly stained and the gland was at the level of epidermal layer (Figure 8).

Stomata
The adaxial epidermis of both A. vasica and A. beddomei were apostomatic (without stomata). The abaxial epidermis were densely stomatiferous in nature. The stomata of both the species were diacytic type and the guard cells were enclosed with two unequal subsidiary  cells with their common walls situated at right angles to long axis of the stoma (Figures 7b and 8c).The guard cells were levelly elliptical in shape and measured 10x25µm and 35x60µm size in A. vasica and A. beddomei, respectively. The abaxial epidermal cells were lobed with their anticlinal walls with thin or thick and slightly wavy. Stomata were densely distributed and they are random in orientation.

Physicochemical analysis
Physicochemical analysis was studied and represented with standard deviation in Table 3. Different physicochemical parameters studied were within permissible limits according to the Pharmacopoeial standards.The total, water soluble and acid insoluble ash of A. vasica and A. beddomei was found to be 12.85, 3.17, 0.95%w/w and 13.20, 2.68, 0.62%w/w, respectively. The water soluble and alcohol soluble extractive were found to be 28.53 and 23.99%w/w for A. vasica and 22.17 and 6.12%w/w for A. beddomei. 5.77 and 7.80% of loss on drying was found in A. vasica and A. beddomei, correspondingly.

Phytochemical screening
The phytochemical examination revealed the presence of various phytoconstituents such as alkaloids, flavonoids, steroids, terpenoids and glycosides in of A. vasica and A. beddomei ( Table 4).

Determination of bioactive contents
The bioactive content of total alkaloid, phenolic, flavonoid, terpenoid, glycoside, sterol, and saponin were determined in the extracts of A. vasica and A. beddomei and the results were depicted in Table 5. The content of alkaloid, phenol, flavonoid, glycoside, sterol and saponins were present in high concentration in A. vasica extracts whereas the terpenoid content were approximately similar in both the plant species.
The alkaloid content of the extracts was expressed as atropine equivalent and it was varied from 3.48mg/g to 238.05mg/g in A. vasica extracts.

HPTLC fingerprinting analysis
HPTLC fingerprint profiling of Adhatoda species were developed with different mobile phases in order to obtain high resolution and peak reproducibility. The phytocompounds were eluted with a mobile phase consisting of hexane, ethyl acetate and formic acid with a ratio of 6:4:0.1v/v/v. Visual examination of obtained chromatogram ( Figure  9) revealed a difference in the chemical composition of various extract of Adhatoda. The fingerprinting analysis revealed the presence of 3, 9, 7 and 5 spots in hexane, chloroform, ethyl acetate and methanolic extracts respectively in A. vasica extract. In case of A. beddomei, 1, 5, 7    Figure 10).

Quantification of Vasicine by RP-HPLC-PDA
Vasicine, a principal bioactive marker has extensively used as a potential bronchodilator. HPLC-PDA method was developed for the estimation of vasicine using a mobile phase of 0.1%v/v trifluoroacetic acid (A) and methanol: acetonitrile (45:40)(B) with a ratio of (70:30%v/v). Linearity graph ( Figure 11) was plotted from the concentration range from 20-120µg/mL with y=29487x-88941 and regression coefficient of 0.998. The retention time of vasicine was found to be 3.946min. The HPLC chromatogram of vasicine standard and different extracts of Adhatoda species were shown in Figure 12.
The hexane, chloroform, ethyl acetate and methanolic extract of A. vasica and A. beddomei revealed that the content of vasicine as 0.143, 0.907, 1.672, 2.051mg/g and 0.042, 0.077, 0.165 and 0.475mg/g, respectively ( Table 6).The high amount of vasicine (2.051mg/g) was found in methanolic extract of A. vasica when compared to other extracts, whereas in methanolic extract of A. beddomei, was found to be 0.475mg/g which was lesser than A. vasica extracts.
The comparative pharmacognostical studies revealed that, the morphological and microscopical differences in the Adhatoda species viz, A. vasica and A. beddomei. There were no crystals seen in A. vasica, where as rod shaped crystals were observed in the leaves of A. beddomei.
Powder microscopic study showed the difference in epidermal trichomes. The presence of non-glandular trichomes were seen in the leaves of A. vasica, which was absent in A. beddomei. Diacytic stomata were seen in both the species of Adhatoda. The total, water soluble and acid insoluble ash of A. vasica was found to be 12.85, 3.17, 0.95%w/w, respectiively. The water soluble and alcohol soluble extractive were found to be 28.53 and 23.99%w/w respectively for A. vasica and loss on drying was found to be 5.77%. A. vasica was found to contain more water soluble, acid insoluble ash and extractive values and A. beddomei was found to contain more total ash and loss on drying.     The preliminary phytochemical screening revealed the presence of more number of phytoconstituents in methanolic extracts of leaves of A. vasica, which was further confirmed by estimation of crude bioactive content. The total alkaloid, phenolic, flavonoid, terpenoid, glycoside and saponin contents were found to be more concentration in polar when compared to non-polar solvents with the order methanol> ethyl acetate> chloroform> hexane, whereas high concentration of steroids was found in non-polar solvents. HPTLC fingerprint profiling revealed that, the chloroform extracts of A. vasica showed a larger number of phytoconstituents when compared to other extracts and also in A. beddomei. HPLC analysis revealed the presence of high content of vasicine in methanolic extracts of A. vasica (2.051mg/g) whereas A.

Adhatoda vasica Adhatoda beddomei
beddomei contains only 0.475mg/g, which was found to be 5 times lesser than the methanolic extracts of A. vasica.

CONCLUSION
Standardization of herbal medicine is a key importance in establishing its proper identity, purity, quality and therapeutic efficacy. The macroscopic, microscopic, physicochemical and phytochemical analysis are the confirmatory test for standardization and quality control. Microscopic, pharmacognostical, preliminary phytochemical screening provides relevant information which may be helpful in authentication of crude drug and checking the adulteration of raw material. Microscopic studies differentiate the difference in the anatomical features and