INTRODUCTION:

Traditional medicine has a long history in India. The Indian Materia Medica contains a wealth of information on folklore practises and traditional aspects of medicinally important natural products¹. Despite the fact that traditional physicians use such plants, many plants have not been scientifically studied.

Nyctanthes arbour-tristis is commonly known as Harsingar, Kalpavriksha, Parijatha, and Night Jasmin and belongs to the Oleaceae family.Harsingar is native to Jammu and Kashmir, the outer Himalayas, east of Assam, Bengal, and Tripura, and south to the Godavari. Traditionally, Seeds of harsingar are used to treat skin diseases including piles and scalp itching infections². The stem of harsingar hasanti-rheumatic potential anti-malaria, and anthelmintics and is used as an expectorant³.

Harsingar leaves are used as colleague, laxative, diaphoretic, in chronic fever and as a diuretic⁴. D-mannitol, β-sitosterole, astragaline, nicotiflorin, oleanolic acid, nyctanthic acid, tannic acid, ascorbic acid, methyl salicylate, carotene, friedeline, lupeol and anthocyanins⁵are the important chemical constituents present in the plant.

MATERIAL AND METHODS:

Plant Collection and Authentication:

Nyctanthes arbour-tristis (Harsingar) leaves are cultivated in Rohtak, Haryana and the same was sent to NIScPR for authentication. Dr. Sunita Garg, Head, Raw Material Herbarium and Museum Division (RHMD) CSIR-National Institute of Science Communication and Policy Research (NIScPR), was identified as Nyctanthes arbour-tristis L under reference number NIScPR/ RHMD/Consult/2021/3951-52-1, 27/12/2021, Pusa Road, New Delhi.

Solvents and Reagents:

Petroleum ether (60-80), chloroform, ethanol, ascorbic acid, gallic acid, Folin Ciocalteu’s reagent, phosphomolybdenum, etc were procured from CDH. All chemicals used in the study were of all analytical grade.

Pharmacognostic evaluation:

Pharmacognostic evaluation provides useful information about the macroscopical, microscopical, and physical properties of crude drugs.

Macroscopic evaluation:

Macroscopic evaluation includes a complete evaluation of plants on the basis of their external features. The plant material is evaluated in terms of shape, size, color, odour, texture and fracture characteristics^6-7.

Microscopic evaluation:

Microscopic evaluation includes powder microscopy and the study of transverse section. In powder microscopy, the powder of the plant was treated with glycerine and chloral hydrate: HCL will be used to mount various characteristics of plants like fibres, starch grain, calcium oxalate crystals, sclereid and many more. In the transverse section, T.S of the plant was mounted with safranin to evaluate characters^8-9.

Physicochemical evaluation:

Standardization parameter includes foaming test, ash values (total ash, water-soluble ash, acid insoluble ash and sulphated ash) loss on drying, extractive value, swelling index crude fibre content and evaluated by standard procedures^10-12.

Preparation of extracts:

Extraction of leaves of N. arbour-tristiswas done by two methods maceration and soxhlation. 500g of coarse powder of leaves of N. arbour-tristiswas weighed and treated with four solvents on basis of polarity (low to high) to get petroleum ether, chloroform, 80% ethanol, and water extract in a successive manner. Total 8 extracts were prepared. These extracts were stored in an airtight container for further use.

Phytochemical analysis:

All extracts were chemically tested for the presence and absence of different phytoconstituents like alkaloids, flavonoids, tannins, glycosides, saponins, proteins and carbohydrates^13-14.

Quantitative analysis:

All extracts were tested for the different classes of phytochemicals present in them like total phenolic content, flavonoid content and tannin content.

Total Phenolic content:

Total phenols present in all extracts viz., Petroleum ether, Chloroform, 80%, hydroalcoholic, and aqueous extract from leaves of N. arbour-tristis was determined using Folin Ciocalteu’s reagent method without any modification^15-16.

Total Tannin content:

Totaltannin present in all extracts viz., Petroleum ether, Chloroform, 80%, hydroalcoholic, and aqueous extract from leaves of N. arbour-tristis was determined using Folin Ciocalteu’s reagent method without any modification^17-18.

Total flavonoid content:

Total flavonoid present in all extracts viz., Petroleum ether, Chloroform, 80%, hydroalcoholic, and aqueousextractfromleaves of N. arbour-tristis was determined using the aluminum chloride without any modification^19-20.

Antimicrobial activity:

Antimicrobial activity of allextractsviz., Petroleum ether, Chloroform, 80%, hydroalcoholic, and aqueous extract from leaves of N. arbour-tristis was determine dusing 96-well microtitration plate method, two-fold dilutions of the antimicrobial agent in a liquid growth medium are placed in smaller proportions in 96-well microtitration plate. After that, a microbial inoculum is added to each well. After thoroughly mixing, the inoculation in a 96-well microtitration plate is incubated (usually without agitation) under appropriate conditions depending on the test microorganism. After 24 to 27 hours of incubation, plates are stained with the Alamar blue dye (resazurin), which is a useful growth indicator^21-23.

RESULTS AND DISCUSSION:

Pharmacognostic evaluation:

The most important and consistent criterion for herbal drug recognition is pharmacognostic evaluation. Pharmacognostic parameters are required for validating the uniqueness, purity, and quality of the crude drug. The comprehensive pharmacognostic evaluation will provide valuable information for the prospect investigation.

Microscopic evaluation of Harsingar:

Different photographs were taken that show Stomates, Gland cell, Vein islet, and Trichome (T). Fig. 1 Representative photomicrographs (x400) of transverse sections of leaves of N. arbour-tristis showing A) Stomates (S), B) Gland cell (GC), C) Vein islet (VI), Trichome (T). Fig. 2 Representative photomicrographs (x400) of Powder microscopyof leaves of N. arbour-tristis showing A) Epidermal layer (EL), B) Bundle of fiber (BF), C) Trichome (T), D) Lower epidermal layer(LDL), E) Cork Cell (CC), F) Upper epidermal layer (UEL).

Fig 1. Represents the transverse sectionof N. arbour-tristisleave A) Stomates (S), B) Gland cell (GC), C) Vein islet (VI), Trichome (T).

Fig 2: Represents the Powder microscopy of N. arbour-tristisleave A) Epidermal layer (EL), B) Bundle of fiber (BF), C) Trichome (T), D) Lower epidermal layer(LDL), E) Cork Cell (CC), F) Upper epidermal layer (UEL).

Plant material standardization:

Table 1: Standardization parameter of N. arbour-tristis

Sr. No	Parameter	N. arbour-tristis (Percentage)
1	Ash value Total ash value	0.73
	Water soluble ash value	0.64
	Acid insoluble ash value	0.3
	Sulphated ash value	0.025
2	Crude fiber content	13.5
3	Extractive value Petroleum ether soluble	4.8
	Alcohol soluble	14.2
	Chloroform water soluble	20
4	Moisture content	4
5	Sweeling index	0
6	Foaming index	More than 100

Phytochemical analysis:

Petroleum ether, chloroform, 80% ethanolic and water extracts of leaves of N. arbour-tristis, were dissolved in respective solvents and tested for the presence of various chemical groups of compounds. Table 2 summarises the results of phytochemical screening.

Total phenolic content:

All the extracts (8) were prepared by different extraction techniques (maceration and soxhlation) by using different solvents like petroleum ether, chloroform, 80% ethanolic, and water extracts of leaves of N. arbour-tristiswere screened for total phenolic content. Amongst all extracts hydroalcoholic extract (Soxhlation) of leaves of N. arbour-tristiscontains maximum phenolic content.

(Table 3).

Table 2: Phytochemical screening of Petroleum ether, chloroform, 80% ethanolic and, water extracts of leaves of N. arbour-tristis

Classes of Phyto-constituent	Name of tests performed	Petroleum extract	Chloroform extract	80%hydroalcoholic extract	Aqueous extract
Carbohydrates	Molisch’s Test Benedict’s Test Fehling’s Test	___	___	++	++
Amino acid	Xanthoproteic Test Ninhydrin Test	__	__	__	___
Steroids	Salkowski’s Test Libermann Burchard’s test	+_	+ +	___	___
Cardic glycoside	Legal’s Test	^¾	^¾	^¾	^¾
Coumarin glycoside	Killer killiani^’s Test	___	___	+	+
Flavonoids	Alkaline Reagent Test Lead acetate Test	+_	+ +	+	+
Phenolic Compound	Ferric chloride Test Liebermann^’s Test Litmus^’s Test	___	___	+ +	++
Alkaloids	Mayer’s Test Wagner’s Test Dragendroff’s Test	___	+ +	+++	+++
Tannin	Gelatine Test	___	+	+	+

Table 3: Total Phenolic content of all 8 extracts of N. arbour-tristis

Sr. No	Extracts	Total Phenolic content (mg GAE/g)
1	Harsingar Maceration Petroleum ether extract (HMP)	2.89±0.058
2	Harsingar Soxhlation Petroleum ether extract (HSP)	4.89±0.013
3	Harsingar Maceration Chloroform extract (HMC)	17.7±0.045
4	Harsingar Soxhlation Chloroform extract (HSC)	15.7±0.057
5	Harsingar Maceration Hydroalcoholic extract (HMHA)	82.7±0.080
6	Harsingar Soxhlation Hydroalcoholic extract (HSHA)	85.2±0.087
7	Harsingar Maceration Aqueous extract(HMA)	67.2±0.069
8	Harsingar Soxhlation Aqueous extract (HSA)	62.2 ±0.034

Total tannin content:

All the extracts (8) were prepared by different extraction techniques (maceration and soxhlation) by using different solvents like petroleum ether, chloroform, 80% ethanolic and water extracts of leaves of N. arbour-tristiswere screened for total tannin content. Amongst all extracts,the aqueous extract (Soxhlation) of leaves of N. arbour-tristis contains maximum tannin content.

Table 4: Total Tannin content of all 8 extracts ofN. arbour-tris

Sr. No	Extracts	Total Tannin content (mg tannic acid/g)
1	Harsingar Maceration Petroleum ether extract (HMP)	5.28±0.009
2	Harsingar Soxhlation Petroleum ether extract (HSP)	5.89±0.18
3	Harsingar Maceration Chloroform extract (HMC)	7.09±0.01
4	Harsingar Soxhlation Chloroform extract (HSC)	7.89±0.043
5	Harsingar Maceration Hydroalcoholic extract (HMHA)	20.75 ±0.067
6	Harsingar Soxhlation Hydroalcoholic extract (HSHA)	25.75±0.089
7	Harsingar Maceration Aqueous extract(HMA)	50.75±0.045
8	Harsingar Soxhlation Aqueous extract (HSA)	53.25±0.069

Total Flavonoid content:

All the extracts (8) were prepared by different extraction techniques (maceration and soxhlation) by using different solvents like petroleum ether, chloroform, 80% ethanolic and water extracts of leaves of N. arbour-tristis were screened for total flavonoid content. Amongst all extracts, the aqueous extract (Soxhlation) of leaves of N. arbour-tristis contains maximum flavonoid content.

Table 5: Total flavonoid content of all 8 extracts of N. arbour-tristis

Sr. No	Extracts	Total Flavonoid content (mg Quercetin/g)
1	Harsingar Maceration Petroleum ether extract (HMP)	2.6±0.05
2	Harsingar Soxhlation Petroleum ether extract (HSP)	2.3±0.045
3	Harsingar Maceration Chloroform extract (HMC)	15.3±0.045
4	Harsingar Soxhlation Chloroform extract (HSC)	12.19±0.078
5	Harsingar Maceration Hydroalcoholic extract (HMHA)	97.5±0.004
6	Harsingar Soxhlation Hydroalcoholic extract (HSHA)	102.4±0.035
7	Harsingar Maceration Aqueous extract(HMA)	33.4±0.045
8	Harsingar Soxhlation Aqueous extract (HSA)	38.7±0.067

Antimicrobial activity:

All 8 extracts were screened for antimicrobial activity against S. typhi, S. aureusB. subtilis, and E. coli using the MIC method, and chloramphenicol was taken as standard. Among all extract, N. arbour-tristis chloroform extract shows maximum antimicrobial potential as shown in table 6 and fig 3.

Fig 3: Represents the antimicrobial activity of different extracts of N. arbour-tristis against selected strains (S. typhi, S. aureus, B. subtilis, E. coli)

Table 6: Antimicrobial potential of all 8 extracts of N. arbour-tristis

Extracts	*S. typhi*	*S. aureus*	*B. subtilis*	*E. coli*
Standard (Chloramphenicol)	62.5	15.6	15.6	31.25
Petroleum ether extract
Harsingar Maceration Petroleum ether extract (HMP)	1000	500	250	250
Harsingar Soxhlation Petroleum ether extract (HSP)	1000	500	250	250
Chloroform extract
Harsingar Maceration Chloroform extract (HMC)	150	125	31.25	62.5
Harsingar Soxhlation Chloroform extract (HSC)	150	125	31.25	62.5
Hydroalcoholic extract
Harsingar Maceration Hydroalcoholic extract (HMHA)	250	250	125	250
Harsingar Soxhlation Hydroalcoholic extract (HSHA)	250	250	125	250
Aqueous extract
Harsingar Maceration Aqueous extract(HMA)	500	500	125	500
Harsingar Soxhlation Aqueous extract (HSA)	500	500	125	500

CONCLUSION:

It can conclude that N. arbour-tristis leaves has antimicrobial potential against S. typhi, S. aureus, B. subtilis, and E.coli. Chloroform extract shows the highest antimicrobial activity against B. subtilis and E.coli. with 31.25 and 62.5 MIC value. Present work suggests that Chloroform extract of N. arbour-tristis can be a good candidate for a natural antimicrobial agent as its show’s activity against both gram-positive and gram-negative microbes.

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Received on 27.01.2023 Modified on 14.06.2023

Research J. Pharm. and Tech 2024; 17(3):1114-1118.

DOI: 10.52711/0974-360X.2024.00174