PHARMACOGNOSTICAL STUDIES, PHYTOCHEMICAL SCREENING AND

THIN LAYER CHROMATOGRAPHY (TLC) PROFILE OF SEED, SEED COAT AND LEAVES OF INGUDI (BALANITES AEGYPTIACA (LINN.) DELILE) WITH SPECIAL REFERENCE TO WOUND

HEALINGSanjeev Kumar,1* Abhishek Kumar,2 Anurag Mishra,3 Neelu Singh,4 Kamal Nayan Dwivedi5

1Research Scholar, 2Associate Professor, 3Research Scholar, 4Research Scholar, 5Professor1,5 Department of Dravyaguna, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi-

221005, India.2 SHEAT College of Pharmacy, Varanasi 221210, India.

3 Department of Physics, Institute of Science, Banaras Hindu University, Varanasi-221005, India.4 School of Environmental Science, JNU, New Delhi, 110067

*Correspondence author; Sanjeev KumarDepartment of Dravyaguna,Faculty of Ayurveda,Institute of Medical Sciences,Banaras Hindu University, Varanasi-221005Contact No.: +91-9451108691 E-mail addresses of first author1; kumarsanjeevdg@bhu.ac.in E-mail addresses of co-authors2;rai.pharma@gmail.com E-mail addresses of co-authors3:mishraanurag.bhu@gmail.comE-mail addresses of co-authors4: nsneelusingh@gmail.comE-mail addresses of co-authors5; kndbhu@gmail.comEmail Id corresponding author’s1*; kumarsanjeevdg@bhu.ac.in

Authors contribution:SK and KND had planned the study design. Physicochemical analysis was done by SK and AK . Phytochemical analysis

was done by SK and AK. AM. SK, NS and KND had contributed in manuscript writing.

Abstract

The present work deals with the preliminary physicochemical, phytochemical and TLC studies on the leaves, seed coat and seeds of Ingudi (Balanites aegyptiaca Linn. Delile). Macroscopical Characters, physico-chemical analysis of dry powder of all three hydroalcoholic extractive sample. The determination of these characters will help future researchers in their Phytochemical analyses of this species. Phytochemical analysis of Hydroalcholic extract of leaves, seed coat and seed extract exhibit the significant anti-inflammatory, antioxidant, antidiabetic, anticancer, antimicrobial and wound healing

activity due to presence of saponin glycosides, tannins, flavanoids and phenolic compound in seed and seed coat along with saponin of leaves. This work will strongly attract both the practioners and researchers in their respective fields.

Keywords: Ingudi, Balanites aegyptiaca Linn. Delile, Phytochemical analysis, antimicrobial, wound healing activity

I Introduction

Ayurveda encloses not only science but religion and philosophy also. We use the word religion to denote beliefs and disciplines contributive toward states of being in which the doors of perception open to all facet of life. In Ayurveda, the whole of life’s journey is considered to be divine. The word philosophy refers to love of truth and in Ayurveda, truth is Being, Pure Existence, The Source of all life. Ayurveda is a science of truth as it is assert in life. From Vedic era to space age, all the researches have been directed by the eminent scholars to achieve the same.

The major limitation of modern medicine, being its undue effects has opened new doors to Ayurvedic system of medicine, where doctors of the traditional systems of medicine are using herbal remedies since many years. In developed and devoloping countries, people are exploring different to modern medicine. In order to make sure the safe use of these medicines, an important step is to setting up standards of quality, safety and efficacious drug for Ayurvedic health care system. Keeping this in mind author made to establish physicochemical standards of the plant Balanites aegyptiaca (Linn) Delile. Balanites aegyptiaca (Linn) Delile (Hindi- Hingen, Hingan, Hingol, Hingot)1. Drug consists of dried fruits of Balanites aegyptiaca (Linn.) Delile (Syn.B.roxburghii Plnach.) family Balanitaceae. The plant is a small tree with glabrous or puberulous branches bearing vary strong sharp ascending spines and bifoliate leaves of two eleptical or obovate, coracious leaflets. It is distributed in the drier parts of the country in western Rajasthan and peninsular India from south east Punjab to West Bengal and Sikkim2.

II ETHNOBOTANICAL STUDIES

The parts of the tree are used as purgative, anthelmintic and expectorant3 (Bhatnagar, L.S. et.al. 1973). The fruits are used as drastic purgative (Bhatnagar, L.S., et. al 1973), skin diseases4 (Pandit, B.R. et.al.1996.); as anthelmintic and purgative5 (Shah G L et. al 1981). The seeds are used in cough (Bhatnagar, L.S., et. al 1973 & Shah G L et. al 1981) and in colic6 (Bhatnagar, L.S., et. al 1973 & Tripathi, Y.C. et. al. 1996). The seeds oil is used in skin diseases7 (Rao, K.P. & Sreeramulu, S.H. 1985); for healing of burns, ulcers and as wormicidal (Bhatnagar, L.S., et. al 1973 & Shah G L et. al 1981). The leaves are used against lice and as antiseptic8 (Katewa, S.S. & Arora, A. 1997) and as and analgesic9 (Kakrani, H.K.N. and Saluja, A.K.1994). The bark is used in skin diseases7

(Rao, K.P. & Sreeramulu, S.H. 1985); urinary disorders, as an anti-inflammatory10 (Maheshwari, J. K., et. al. 1986).) Purgative and anthelmintic ( Shah G L et. al 1981 & Tripathi, Y.C. et. al. 1996). The root is used as anthelmintic11 (Joshi, P 1991); anti dysenteric, analgesic, antiulcerogenic, alexipharmic, in leucoderma, skin diseases6 (Tripathi, Y.C. et. al. 1996) and as antiseptic antiseptic (Katewa, S.S. & Arora, A. 1997) and as and analgesic (Kakrani, H.K.N. and Saluja, A.K.1994).

III SOURCE OF DRUG

Drug was collected from rural area of Etawah district (latitude 26.8117° N, longitudinal 79.0047° E), U.P and identified by the teacher of Dravyaguna department in faculty of Ayurveda B.H.U. Accession no. of my raw drug is DG/18-19/194 (dry fruit).

IV MACROSCOPIC CHARACTERSTIC OF LEAVES, SEED COAT AND SEED POWDER OF BALANITES AEGYPTIACA LINN. DELILE.

Physico-chemical evaluation

Materials: 1. Powdered leaves, seed coat and seeds of Ingudi (Balanites aegyptiaca.Linn.Delile).

2. Petri dish.

Method: 5 gm powder was taken in a Petri dish and examined with naked eye.

Phytochemical screening

Materials: 1. Powder of Ingudi (Balanites aegyptiaca.Linn.Delile).

2. Phloroglucinol

3. Conc. HCl

4. Microscope

5. Glycerine

6. Slide & Coverslip

7. Watch glass

Method:

5 gm powder of all three samples (leaves, seed coat, seeds) was boiled separately with chloral hydrate solution in small quantity. Cleaved powder was removed in three separate watch glasses respectively and stained with one drop each of phloroglucinol and conc. HCL. A little of the treated powder was mounted in dilute glycerin and the slide was observed under microscope at low power. The morphological studies and the analysis of the seed powder revealed that, it is creamish white in color with aromatic odour.

Table1: Physiochemical parameters of Ingudi (Balanites aegyptiaca(Linn.) Delile) leaves

S. No. Parameters ObservationsLeaves

I. Physical TestsNature Dark scabrousColor GreenOdour Odourless

Taste BitterShape & SizeTexture Smooth

II. Extractive Value (%w/w)Hydroalcoholic 9.65

III. Loss on Drying (%w/w)

6.93

IV. Ash values (%w/w)Total ash 8.7Acid insoluble ash 3.5Water soluble ash 6.3

V. Foreign Matter NILTable2: Physiochemical parameters of Balanites aegyptiaca(Linn.) Delile (Ingudi) Seed coat

S. No. Parameters ObservationsBark

I. Physical TestsNature Dark scabrousColor Brownish Odour OdourlessTaste BitterShape & SizeTexture Rough

II. Extractive Value (%w/w)Hydroalcoholic 32

III. Loss on Drying (%w/w)

4

IV. Ash values (%w/w)Total ash 8Acid insoluble ash 5Water soluble ash 3

V. Foreign Matter NILTable 3: Physiochemical parameters of Ingudi (Balanites aegyptiaca(Linn.) Delile) Seed

S.no Parameters Observation Seeds

I Physical tests Nature Coarse powder

Colour Creemish white Odour Aromatic Taste Bitter

II Extractive value (% w/w) Pet. ether 2

Hydro-alcohol 1.1 Aqueous ------------------

III Loss on drying (%w/w) 9IV Foreign matter Nil V Fluorescence No fluorescenceVI Ash value (% w/w)

Total ash 4.5 Acid insoluble ash 6.5 Water insoluble ash 2

V PHYTOCHEMICAL SCREENING

Qualitative examination of inorganic matters & determination of heavy metals was done as per reported methods. The dried powdered was subjected to preliminary phytochemical screening for qualitative detection of phytoconstituents of all the three samples. For further studies extraction was done by soxhlet apparatus respectively. The shade dried samples of Ingudi (Balanites aegyptiaca .Linn.Delile) family Simarubaceae ) were reduced to fine powder (#40 size mesh) and around 45 gm of powder was subjected to successive hot continuous extraction (soxhlet) with petroleum ether ( 60- 800C), ethanol and distilled water. Each time before extracting with next solvent the powdered material was air dried in hot air oven below 500C. After the effective extraction, the solvent was distilled off, the extract was then concentrated on water bath and the extract obtained with each solvent was weighed. Its percentage was calculated in terms of air dried weight of plant material, Color and consistency of the extract was noted.

VI RESULTS

Macroscopic evaluation

The Macroscopic characters found are discussed in (Table 1, 2, 3).

External morphology of leaves, seed coat and seeds of Balanites aegyptiaca linn. Delile

External morphology of leaves, seed coat and seeds of Balanites aegyptiaca Linn.Delile are shown in

Phytochemical screening

The results demonstrated presence of Saponins glycosides, alkaloids, tanins and flavanoids in seed

coat and seeds but not in leaves of Ingudi (Balanites aegyptiaca (Linn.) Delile). In leaves only saponin

glycosides are present. Cardiac glycosides and coumarins are absent in all three samples. Saponin

glycosides in all the samples, carbohydrate present only in seed coat shown in (Table 1, 2, 3)

VII THIN LAYER CHROMATOGRAPHY

Pharmacopoeias are increasingly insisting to employ chromatography as a mean for evaluating quality

and purity. Principle of chromatographic separation: Different distribution of the analysts between

mobile and stationary phase results in different migration velocities.

Chromatography is the collective term for a set of laboratory techniques for the separation of

mixtures. The mixture is dissolved in a fluid called the mobile phase, which carries it through a

structure holding another material called the stationary phase. The various constituents of the mixture

travel at different speeds, causing them to separate. The separation is based on differential partitioning

between the mobile and stationary phases.

Distance travelled by the solute

RF Value=

Distance travelled by the solvent front

In any chromatographic technique, a stationary phase (usually a solid, thick liquid, or bonded

coating) stays fixed in one place, and a mobile phase or eluent (usually a liquid or gas) moves

through it or across it.

VIII POWDER MICROSCOPY OF SEED COAT AND SEED POWDER

DISCUSSION

In phytochemical study of seed of Ingudi shows presence of Proteins, Amino acids, Fat & Oils, Volatile Oils, Saponin Glycosides, Tannins & Phenolic Compounds, seed coat containg Saponin Glycosides, Tannins & Phenolic Compounds, and flavanoids and leaves containg basically saponins it may be further helps in identification of plant (Table 5). Hydroalcholic leaves, seed coat and seed extract showed the significant activity against the microbes as hydro-alcoholic extract mainly extracts the phenolic component of the plant which is mainly responsible for the biological activity. It was reported that phenolic compounds exert antioxidant, antidiabetic, and anticancer effect etc12,13,14. Flavonoids to promote the wound healing process mainly due to their astringent and antimicrobial property, which seems to be responsible for wound contraction and increased rate of epithelialization. Similar types of wound healing activity were reported on Ingudi15. The flavonoid fraction significantly increased the skin breaking strength and hydroxyproline content which was a reflection of increased collagen levels by increased cross linking of collagen fibres. In addition, increase in dry granulation tissue weight indicated the presence of higher protein content16. Flavonoids exhibit many pharmacological activity due to their anti-inflammatory, anti-fungal, antioxidant and wound healing properties, reduce lipid peroxidation not only by preventing the inception of cell necrosis but also by improving vascularity17,18,19.Tannin is one of the important plant constituent responsible for the wound healing mainly due to astringent and antimicrobial property20. This is very big clue for newer researchers to prove that the Ingudi seed and seed coat exhibit anti-inflammatory, antioxidant, anti-diabetic, anticancer, antimicrobial and wound healing activity.

Table 4: Showing Rf value of different parts of Ingudi (Balanites aegyptiaca Linn. Delile)21

S. No Name of parts used in Extract Rf Value1 Hydro alcoholic extract of Leaves 0.712 Hydro alcoholic extract of Seed Coat 0.6, 0.93 Hydro alcoholic extract of Seed powder 0.8

Table 5: Phytochemical screening of Hydroalcoholic Extracts of different parts of Ingudi (Balanites aegyptiaca(Linn.) Delile)

IX CONCLUSION In present study phytochemical standards have been established with regards to leaves, seed coat and seeds of Ingudi (Balanites aegyptiaca Linn.Delile, Local name Hingot). The phytochemical investigation shows the presence of Saponin glycosides, Volatile oils, Proteins, Amino Acid, Fat & Oils, Steroids, Flavanoids, Tannins and Phenolic Compounds in the seeds, Saponin glycosides, Flavanoids, Tannins and Phenolic Compounds in seed coat and saponin of leaves of Balanites aegyptiaca. Study was carried out in order to assess the quality of seeds of Balanites aegyptiaca Linn. Delile and also to detect the adulteration and substitution etc., which may be helpful to researchers in future.

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