Pharmacognostical, Phytochemical and Gas Chromatography Mass Spectroscopy Profiling of Stenosiphonium russellianum Nees

Stenosiphonium russellianum Nees. is rarely known species belongs to the family Acanthaceae and it is a shrub, found above 500m on slopes of mountain. It was traditionally used for wound healing in and as blood purifier. The current study designed to provide the requisite pharmacognostical and phytochemical properties of Stenosiphonium russellianum. Pharmacognostical studies like microscopic and macroscopic analysis of the leaves were carried out. Physiochemical parameter and preliminary phytochemical screening for secondary metabolite were also performed. Extracts were taken from nonpolar to polar solvants like hexane, diethyl ether, ethyl acetate, alcohol and water. Their extractive values are calculated. GCMS analysis of hexane, diethyl ether, ethyl acetate and ethanol extract of the leaves of Stenosiphonium russellianum were studied. Preliminary phytochemical evaluation showed the presence of alkaloids, phytosterols and glycosides. GCMS analysis revealed the presence compounds like lupeol, gamma sitosterol and stigmasterols. In conclusion, the information obtained from these studies can be used Original Research Article Mahalakshmi et al.; JPRI, 33(46B): 95-104, 2021; Article no.JPRI.74998 96 as markers in the identification and standardization of this plant as an herbal remedy and also towards further pharmacological activity estimation.


INTRODUCTION
Acanthaceae. Acanthaceae is dicotyledonous family consisting of 250 genera and about 2500 species. Leaves, stem, flowers and roots of most of the species of Acanthaceae has pharmacological activities such as wound healing, anticancer, antioxidant, Antifungal, antiviral, anti-inflammatory, antipyretic, antioxidant, insecticidal, hepatoprotective, antiplatelet and immunomodulatory. The leaves are the most often used part in the Acanthaceae family and specially used for wound healing. These pharmacological actions are produced by presence of phytochemicals like alkaloids, phytosterols, glycosides, flavonoids, phenolic compounds, triterpinoids, benzonoids and naphthoquinones present in the species of the family [1].
Stenosiphonium Nees (Acanthaceae) was one of the genera described by Nees. This genus forms a well-defined and putatively monophyletic group, distributed in southern India and Sri Lanka, which were morphologically similar to strobilanthes Blume [2]. Stenosiphonium russellianum is characterized by leaves with silky undersurface, stems not winged, corolla blue to violet and has tube ventrilose usually found in 500m above the slope of the hills [3]. Regional names are kal-kurinji and karumaththi-poondu. For wound healing, the leaf paste of Stenosiphonium russellianum is applied in the morning for two days [4]. It is also used as blood purifier [5].
As we know that plant species consumed by animals are known to have therapeutic effect on human also. For example, Cynodon Dactylon (scutch grass), Abutilon indicum (Indian mallow leaves), Solanum trilobatum (pea eggplant), Hibicus rosasinensis, these are known to produce many pharmacological activity which were consumed by animal. With all this background, we choose this plant for research. In this article you can get a broad detail regarding the monographs and phytocompounds of the species Stenosiphonium russellianum.

Plant Material Collection and Authentication
The leaves of Stenosiphonium russellianum were collected from hills at village Thevanadhapettai, Gingee district, Tamil Nadu, India. The plant material was identified and authentication by Dr. N. Ayyappan, Researcher, French Institute of Pondicherry, Puducherry, India. Leaves were then washed to remove adhering material, shade dried and powdered. The powders were stored in an airtight self-sealed cover.

Morphological characters
The macroscopic study was conducted to aid in the identification as well as standardization of this plant species. The fresh leaves were subjected to macroscopic studies which comprised of organoleptic characters. Morphological studies of leaf such as color, size, odor, taste, surface characteristic were examined using the terms and outlined given in [6].

Microscopic analysis
For microscopic studies of the fresh leaves, freehand sections of midrib with lateral extensions of lamina on either side of the leaves were taken to prepare the specimens, stained with phloroglucinol in Hcl and mounted with glycerin for microscopic evaluation [6,7]. Sections were viewed under 10x, and 45x magnifications in microscope for the identification of various regions and photographs were taken.

Physicochemical analysis
Physiochemical parameter such as loss on drying of leaves, loss on drying of powder, total ash, and acid insoluble ash of leaves powder of Stenosiphonium russellianum were performed according to the WHO guidelines on quality control methods for medicinal plant material [8,9].

Fluorescence analysis
The fluorescence characters of powdered drugs of medicinal plants helps in the determination of quality and purity of test samples. To study the fluorescence behavior powder of Stenosiponium russellianum were leaves treated with few drops of different reagents on a clean watch glass, waited for few minutes and observed under UV Visible at 254nm [10,11].

Preparation of leaves extract and their extractive value
250gms of coarsely powdered drug at room temperature materials of Stenosiphonium russellianum is placed in a 500 ml stoppered conical flask. 500 ml of five different solvents of from highly non polar to polar (hexane, diethyl ether, ethyl acetate, ethanol and water) were poured on top until completely covered the drug material and kept aside for 7 days with periodical shaking. At the end, micelle of Stenosiphonium russellianum is separated from marc by filtration and then solvents are removed using distillation. Percentages of extractive value of extracts were calculated. The obtained extracts of hexane, diethyl ether, ethyl acetate, ethanol and water of Stenosiphonium russellianum were stored in airtight glass container for further phytochemical and pharmacological analysis [12][13][14].

GC-MS Analysis
The Clarus 680 GC was used in the analysis employed a fused silica column, packed with Elite-5MS (5% biphenyl 95% dimethylpolysiloxane, 30 m × 0.25 mm ID × 250μm df) and the components of extracts were separated using Helium as carrier gas at a constant flow of 1 ml/min. The injector temperature was set at 260°C during the chromatographic run. The 1μL of extracts of S. russellianum injected into the instrument the oven temperature was as follows: 60 °C (2 min); followed by 300 °C at the rate of 10 °C min−1; and 300 °C, where it was held for 6 min.

RESULTS AND DISCUSSION
Authentication and standardization are the main prerequisite steps when come for research on natural products in any system of medicine [18]. The pharmacognostic studies are the identity of crude drugs; it gives a complete characterization of the species.

Morphological Characters
Macroscopic as well as organoleptic evaluation of the leaves includes position and arrangement, size, shape, base, texture, margin, apex, veination, colour, odour, taste of leaves were observed and listed in Table 1.

Microscopical Studies
Transverse section of leaf midrib shows rounded shape with single layer of the adaxial and abaxial epidermis with small trichomes. Midrib parenchymatous cells are 7 layers of rounded closely arranged. Vascular bundles are C shaped open collateral. More than 16-18 xylem rows in vascular bundles. Phloem cells are present in abaxial side. Leaf lamina projection are connected with midrib (Fig. 1). Adaxial epidermis consists of uniseriate conical trichome which might be eglandular, multicellular and uniseriate unbranched. Abxial epidermis consists of unicellular conical trichome and Simple filiform trichome, these might be eglandular, multicellular and uniseriate unbranched. Epidermal layer has abundant diacytic stomata with cystoliths (Fig. 2).

Physicochemical Analysis
Physicochemical parameters like loss on drying of leaves, loss on drying of leaves powder, total ash and acid insoluble ash of powder were investigated and the results were interpreted for determining the values and are summarized in Table  2.
Physicochemical parameters identification of a crude drug is a crucial factor for proper identification of plant.

Fluorescence Analysis
Different reagents treated powder was observed at UV 254nm and visible light for fluorescence characteristics. Those observations are presented in Table 4.

Extractive Value
Extractive values of all the solvent extracts shown in Table 3. Ethanol soluble extractive value was found to be more than ethyl acetate soluble extractive value however it was less than aqueous soluble extractive value.

Preliminary Phytochemical Analysis
Name of the test and its inference of preliminary phytochemical screening of the extracts of leaves were interpreted in the table 5. Phytochemical investigation of extracts of Stenosiphonium russellianum revealed the presence of alkaloids, glycosides, flavonoids, phytosterols, phenolic compounds and tannins. Among various solvent used for extraction, ethyl acetate extracts of leaves gave maximum positive results. Alkaloids are present in ethanol and aqueous extract whereas glycosides are present only in ethyl acetate extract. Hexane and diethyl ether extracts didn't showed any positive inference in all performed test. Phytochemical analysis of the leaves extracts revealed the presence of constituents known to exhibit therapeutic as well as physiological activities.

CONCLUSION
The pharmacognostic standards for the leaves of Stenosiphonium russellianum are laid down for the first time. Morphological and microscopic studies of leaves will enable to identify the crude drug. Preliminary phytochemical screening as well as GCMS profiling will be useful in further research on this species like isolation of lead and determination of pharmacological activity.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.

NOTE
The study highlights the efficacy of "herbal remedy" which is an ancient tradition, used in some parts of India. This ancient concept should be carefully evaluated in the light of modern medical science and can be utilized partially if found suitable.