Macroscopic, Microscopic, Preliminary Analytical Evaluation and Quality Assessment of The Seeds of Quercus Leucotrichophora

The Banj oak or Ban tree, Quercus leucotrichophora, belongs to the Fagaceae family. Banj trees are evergreen and can grow up to 40 meters in height.[1] The Banj tree may grow up to 2400 meters above sea level in the Himalayas. There are several banj trees in the Shimla, Kullu-Manali, and upper Mandi regions of Himachal Pradesh.[2]

Seedlings can survive in some shade for the first two years of their lives, but beyond that, the tree needs moderate to full sunshine to grow. It does not thrive in arid areas and grows best in moist, well-drained soils made of shale and clay loams. Depending on where you live, mature seed may remain viable for months after it has ripened.[3] The canopy of this tree, Quercus leucotrichophora, A. Camus, is broad and elliptical. As trees grow older, the bark changes from being smooth and tan-brown to being somewhat corky and wrinkled.[4]The leaves are elongated ovals with sharp teeth, placed alternately.[5] The name "leucotrichophora" (which means "which has white hairs") stems from the fact that when the leaves grow, the upper surface turns brilliant green while the bottom surface, which has white hairs, turns silvery grey.[6] While flowers (female inflorescences) are found near the base of the leaves, catkins (male inflorescences) are found at the top of the branches.[2]The tree's gum has long been used to cure gastrointestinal problems and gonorrhoea, especially in children. The seeds' astringent and diuretic properties are used by people to treat asthma, diarrhoea, and indigestion.[7]

MATERIALS AND METHODS

Collection, Authentication and Preservation of the sample

The plant specimen was gathered in its natural setting at Chail-chowk in Mandi, Himachal Pradesh. Dr. Pankaj Sharma, Senior Scientific Professional Himachal Pradesh State Biodiversity Board, Shimla, Himachal Pradesh 171001, verified the plant's taxonomic identification. HPSBB/ - 4109, dated 14 January 2022.[8]

Preparation of plant extract

Extraction: Extraction in science is a partition cycle used to isolate a substance from a grid. As of late dynamic standards from plants have been confined or gotten as refined results of exactly known power and soundness. The plant contains compound substances some of which give help from different sick circumstances. Menstruum is the solvent used for extraction and Marc is the undissolved residue left over from the process.[9]

Selection of solvents:

To decide the extraction values, Quercus leucotrichophora was separated with seven solvents as indicated by their extremity: petroleum ether, toluene, chloroform, ethyl acetate, acetone, methanol and water.  Methanol is a semi-polar dissolvable that can remove numerous phytoconstituents and water is a polar dissolvable that is modest and non-poisonous.[10]

Procedure

A porous bag or "thimble" produced from a sturdy filter paper was filled with 20g of dried QL seed powder after being precisely weighed, and it was then placed in the Soxhlet apparatus' thimble chamber. The heating mantle is used to warm the extraction solvent which is placed in the flask with a round bottom. The extraction solvent used determines the heating temperature. The bottom flask's solvent vaporizes into the condenser as a result of heat, and then drips back into the sample thimble. The liquid content is drained into the bottom flask once more when it reaches the siphon arm, and the clear solution in the siphon indicates that the operation is complete.

The filtrate is transferred to a flat-bottomed dish that has been pre-weighed, evaporated to dryness on a water bath and then dried completely in an oven for six hours. The dish is then cooled by placing it in a desiccator for 30 minutes and it is immediately weighed. This process is repeated with all seven solvents. The content of the extractable matter is calculated.[11]

MACROSCOPIC STUDY

Morphological characteristics

Morphological characteristics of seeds of Quercus leucotrichophora such as shape, size, texture and colour were studied as per visual observations and verifying with taxonomical standard books for authentication.  [12]

Organoleptic study

The organoleptic properties as odour, colour, taste and touch of Quercus leucotrichophora seed powder were recorded and documented via visual and sensory observations.[13]

MICROSCOPIC STUDY

Microscopy of transverse sections

The seeds were soaked in water for 24hours and then free-hand thin transverse sections of seeds were taken. These transverse sections were stained with safranin following the standard prescribed methods.

Powder microscopy

Seed powder (14#) was taken in a glass slide mounted with glycerine and was examined under compound microscope.[14]

PHYSICOCHEMICAL ANALYSIS

Assessment of various parameters such as moisture content, loss on drying, ash value, sulphated ash value, water-soluble ash value, acid-insoluble ash value, alcohol soluble extractive value, water-soluble extractive value and photoluminescence studies following standard procedures that are recommended by Ayurvedic Pharmacopoeia of India (API) and other standard texts.[15]

THIN LAYER CHROMATOGRAPHY

Preparation of stationary phase

Sample was applied 1cm above from the baseline on TLC-plate precoated with 0.25 mm layer of silica gel GF 254 with fluorescent indicator (Merck’s) of size 8*2 cm.

Development of solvent system

Among a number of solvents, the best mobile phase was found. The solvent system water and methanol (1:1) gave maximum number of spots and best resolution.

Development of chromatogram

Chromatogram was developed using glass container which was pre-saturated using mobile phase i.e., water and methanol solution. The sample loaded TLC plate was placed in chromatogram such that mobile phase remains below the sample application point. The sample elution occurs due to capillary action.[16]

Scanning and detection of spots

The developed TLC-plate was air-dried. The spots were visualized under UV366nm and Rf was calculated using following formula:

Rf = Distance travelled by spot from origin/Distance of Solvent front

All the values were calculated and documented.[17]

RESULTS AND DISCUSSION

MACROSCOPIC STUDY

Morphological characteristics

The seed of Quercus leucotrichophora have two surfaces. The outer surface i.e., a dark brown acorn cap which is hard and rough in appearance. It has soft nut-shaped inner surface which is white in colour before drying but becomes blackish brown after drying.

Figure 2: QL Seed with Acorn Cap

Organoleptic study

Table1: Organoleptic properties of Quercus leucotrichophora seed

MICROSCOPIC STUDY

Transverse section

The cross-section of the seed showed (Figure 3) that ithadadicotyledon which is made up of a plumule, a radical, a cotyledon, an endosperm, parenchymatous cells, and atigellum. Plumule (shoot apical meristem) and Testa (root apical meristem) are made up of parenchymatous cells which form the stem and root when the seed germinates. Seed coat was thick, had one or two layers, and looked yellowish brown. Epidermis was attached to the inside of the Testa and looked like a single layer. Endosperm was made up of oval or circular cells with thick walls that were closely attached to each other. Parenchymatous cells are simple, oval, and round, and they are all packed together. There were parenchymatous cells inside the endosperm, and the thick cotyledon was attached to an axis called the tigellum. The tigellium had a plumule that stuck out and a root was hidden between the cotyledons.

Figure 3: T.S. of Seed of Quercus leucotrichophora

Powdered microscopy

Seed powder is darkish brown. It has a characteristic odour and slightly bitter in taste. Beneath the microscope it showed interlocking tracheid, dead angular cells with solid hard walls, wide lamps, and narrow end walls. Tightness inserted as a whirl or helix with wind tracheid.

The stairways seem to thicken with scalariform tracheid because they are placed on the ground in the form of transverse bands.

Figure 4: Powder microscopy of powdered seeds of Quercus leucotrichophora

PRELIMINARY PHYSICOCHEMICAL ANALYSIS

The weight of whole fruit was found to be 34.02±4.53 whereas; the weight of seed was 9.34±0.23. After drying and size reduction the power was of Blackish Yellow colour having moisture content7.233±0.0283%. The ash value determines the quantity of the inorganic material present in the drug which was found to be within the limits in present study. The average percentage of total ash of seeds of Quercus leucotrichophora is 21.449 ± 2.629%w/w. This may be due to presence of high amount of iron (Fe) and zinc (Zn) and Calcium (Ca). The acid insoluble part of ash was found 0.986% w/w whereas water soluble part was18.746%w/w. The extractive values for various solvents such as methanol, n- hexane, chloroform, ethyl acetate, n-butanol and aqueous were found to be 18.746, 4.6224, 0.294, 0.4982, 0.938 and 3.9834 respectively. Swelling index was measured to be 8ml/g. The results of preliminary physicochemical analysis are given in Table 2.

Photoluminescence studies

The powdered drugs when subjected to ultraviolet light and visible light in the presence of various chemical reagents, exhibit characteristic fluorescence.

The obtained results are mentioned in Table3.

Table 3:  Fluorescence characteristics of Quercus leucotrichophora seed extracts

Various phytochemical analysis tests supported   that the methanolic extracts contain alkaloids, carbohydrates, flavonoids, phenolic compounds, tannins and glycosides.

The results of the performed tests are depicted in Table4.

Table 4: Qualitative analysis of Quercus leucotrichophora seed in methanol extract

    + = Present, - =Absent

Qualitative Chromatography of methanol extract and ethyl acetate extract was carried out by using Methanol (MeOH): Distilled water (DW) (2:3) and MeOH + Di-Chloro Methane (DCM) (8.5:1.5) solvent systems simultaneously to assure the presence of various phytochemicals. The chromatogram of methanol extract showed a total of 5 spots at different Rf values. Ethyl acetate extract showed 3 spots having different Rf values.

The Rf values of the obtained spots is calculated and is mentioned in Table 5.

Table 5: Thin layer chromatographic data of Quercus leucotrichophora seeds.