1. INTRODUCTION:

About 80% of the generation of the world is mainly dependent on the agricultural field of plants, crude extract and their aromatic oil. Crude extracts and essential oil have owning therapeutic potential for human health. Medicinal plants that contain therapeutic potential have a large number of therapeutic compounds used to cure different diseases. These therapeutic compounds have a valuable source of antioxidants. It contains many secondary metabolites. These secondary metabolites are aromatic substances that are used as protective molecules for mankind. Itis a new origin of antimicrobial evaluation and impacts the absorption, digestion etc. of the human body. Many plants have both therapeutic and nutritional functions that are cost-effective, have no side effects, and are easily available for prolonged period use^1,2.

Covering all these facts, researchers need to invent noble and new remedies from biological factors. Therefore, most indigenous medicinal plants, Helicteres isora need to be investigating for its medicinal use. It is the main ayurvedic herb possessing therapeutic and nutritional value. It is present in the forest and high hills as a small tree and sub-deciduous shrub with cogged margin, hairy leaves commonly called in English “East India screw tree” and Hindi “Marorphali”³. Every part of this plant is used therapeutically in Indian system of medicine (ISM) for different diseases such as diabetes, gastric problems, hemorrhages, diarrhoea, wounds, dysentery and ulcer infections. Fruit of this plant is present in form of twisted like appearances throughout India, applied in many zones for therapeutic purposes. The bark of this plant is used in diabetes and emphysema. Leaves of this plant is used for treatment of wound and snake bite. Different types of bioactive compounds such as sitosterol, isorin, betulic acid, isocucurbitacin B and cucurbitacin B were isolated from the root of Helicteres isora. This type of compound was studied by pharmacokinetics for drug likeness properties. Moreover, this plant has various bioactive potentials such as antimicrobial, anticancer, hepatoprotectic, antioxidant, antidiabetic, hypolipidemic and antidiarrhoeal activity⁴. Similarly, other plants Jatrophacurcas, Boswellia serrate, Vitex negundo have been good antimicrobial potential in therapeutic condition. Eupatorium also obtained anti-inflammatory as well as wound healing activity^5,6. Every part of plants is used for antimicrobial activity which has been reported by kumar et al and Shanmuganathan et al^7,8. This work, therefore, was designed to investigate antimicrobial, GCMS analysis, drug likeness properties of Helicteres isora on the basis of phytochemical screening. The main concept of this article is give outcomes of several experimental data focusing on the different extract and essential oil of plant for therapeutic uses.

2. MATERIALS AND METHODS:

2.1 Accumulation and identification of plant material:

The fruits of H. isora were accumulated from Rajajinational park, Haridwar, Uttarakhand, India. This zone seizes up the 820 km² area and have coordinates between 29°56' N- 78°22' E. Complete plant ready with a herbarium sheet and sent to the Dehradun Botanical survey of India (BSI) for future identification. Plant specimen was recognized from BSI Dehradun on basis of the already present database and morphological features. A voucher specimen was given in form of Accession No. 180 and one file of the herbarium was deposited for future references in BSI, Dehradun.

2.2 Test pathogens:

Bacterial strains Klebsiella pneumoniae (MTCC 4030), Pseudomonas aeruginosa (MTCC 2474), Streptococcus pyogenes (MTCC 442), Escherichia coli (MTCC 40) and Staphylococcus aureus (MTCC 1144) were acquired from the Institute of Microbial Technology (IMTECH), Chandigarh, India.

2.3 Extraction of crude extract and essential oil:

The fruits of H. isora were washed under running tap water and air-dried to remove the moisture. Dried fruits of this plant were scrunched with the help of a mortar pestle and then grinded with the electric grinder (Electrocom). For the crude extract preparation, 100g of dried sample was kept in Soxhlet and immersed sequentially in different solvents like petroleum ether, methanol, ethanol and water. The temperature of all was set according to the solvents for 3days complete extraction. After that the extract was separated by the Whatman filter paper and the solvent was evaporated by Rota evaporator. Similarly, the essential oil was extracted by the Hydro-distillation process in which the dried sample was immersed in distilled water and oil was collected into the Clevenger apparatus. This oil from Clevenger was separatedwith the help of a separating funnel. Oil was mixed with hexane because it actsmiscible substance for essential oil and stored at 4°C airtight for further processing.

2.4 Phytochemical screening of fruit extract of the plant:

Phytochemical screening of different solvents such as petroleum ether, methanol, ethanol and water was performed by using the standard procedure⁹.

2.5 GC-MS and Computational analysis of ADME Prediction:

Essential oil of H. isora was characterized by using Thermo Scientific TSQ 8000 GC-MS from central instrumentation library (CIL), Punjab University, Chandigarh, India. Property of ADME (absorption, distribution, metabolism and excretion) wasanalyzed by using pharmacokinetic study within the ADME algorithm inbuilt with the pharmacokinetic study¹⁰. ADME are inbuilt in the ADME algorithm software package. Absorption of drugs depends on various factors like water solubility, skin permeation and P-Glycoprotein substrate. Now, the predictions of metabolism are based on different inhibitors of CYP. All the above predicted/expected value are in parallel to the standard ranges.

2.6 Antimicrobial activity (Antagonistic study of indicator pathogens):

The antimicrobial efficacy of H. isora was determined by the agar well diffusion method¹¹. Cells from stock cultures were transferred directly to Muller Hinton broth sequentially and all pathogens were incubated at 37°C, for antimicrobial susceptibility testing. 0.5 McFarland standard (1.5 ×10⁸ CFU/ml) of all bacterial strains was prepared. The 50mg/ml final concentration of all plant extracts and 50μl/ml essential oil was prepared in dimethyl sulfoxide (DMSO). Antibiotics (clindamycin and ciprofloxacin) were used as a positive control. Muller Hinton agar media plates were prepared for antimicrobial testing. Solidified media plates of Muller Hinton were tested against different bacterial strains. 100μl of bacterial culture was spread on plates, 45 μl sample was dispensed into 6mm diameter well after that all plates were incubated at 37°C for 24 hours. All experiments were performed in a triplicate manner under aseptic conditions. Thus, all results were measured as a zone of inhibition and the mean value of the zone of inhibitionwas calculated in a triplicate manner.

2.7 Statistical analysis:

All experiments were tested statisticallyby one-way analysis of variance (ANOVA). These experiments were analyzed in a triplicates manner, exhibited as mean±SE (Standard error). The variation between means was determined at P≤0.05 for significant value using Microsoft Excel 2010.

3. RESULTS:

3.1 Phytochemical screening:

Phytochemical testing of fruit extract of H. isora showed the presence of different phytoconstituents i.e., carbohydrate, phenol, alkaloid, saponins, flavonoids which were mentioned below (Table 1).

Table 1: Phytochemical screening of fruit extract of Helicteres isora

Phyto- chemicals	Extracts
Phyto- chemicals	Petroleum ether	Methanol	Ethanol	Water
Alkaloid	+	+	-	-
Glycoside	+	-	-	-
Carbohydrate	+	+	+	-
Flavonoid	+	+	+	-
Phenol	+	+	-	+
Saponin	-	+	+	+
Terpenoid	-	-	-	+
Oxalate	-	-	-	-
Quinone	-	+	-	-

+ve: positive, -ve: negative

3.2 Antimicrobial evaluation:

Antimicrobial screening of different extracts of fruit of H. isora examined on the basis of absence or presence of diameter against different pathogens. The outcomes of antimicrobial activity of crude extracts, essential oil and reference drugs were shown in figure 3. The graph showed a large variation of diameter between different extracts, oil and control. It was seen that Pseudomonas aeruginosa is the dominant pathogen among all four crude extracts and methanol was the most dynamic extract followed by the ethanol, water and petroleum ether. Furthermore, in essential oil, Escherichia coli is the most sensitive pathogen followed by tested pathogens Klebsiella pneumoniae, Streptococcus pyogenes and Streptococcus aureus. Various drugs are used for sensitivity testing. Escherichia coli exhibited the highest inhibition in ciprofloxacin followed by clindamycin.

Fig 1:Chromatogram of GCMS of essential oil of Helicteres isora

Table 2: Composition of GCMS of different compounds of Helicteres isora essential oil

Plants	S No.	Compounds	Formula	Molecular weight (g/mol)	Retention time
*Helicteres* *isora*	1	6,10,14-Trimethylpentadecan-2	C₁₈H₃₆O	268.48	17.73
	2	n-Hexadecanoic acid	C₁₀H₂₀O₂	172.26	19.64
	3	Phytol and isomenthol	C₂₀H₄₀O, C₁₀H₂₀O	296.53, 156.26	21.84

Table 3: Physiochemical properties of attained GCMS compounds 1-3a&b

Compound No.	nROTB	HBA	HBD	iLogP	TPSA	In silico % absorption
1	12	1	0	4,39	17.07	102
2	8	2	1	2.50	37.30	95.13
3a	13	1	1	4.71	20.23	101.02
3b	1	1	1	2.55	20.23	101.02

nROTB: number of rotation bonds, HBA: H-bond acceptor, HBD: H-bond donor, iLogP: lipophilicity, TPSA: polar surface area (Topological)

Fig 2: Egg boiled predictions of compounds 1-3a &b

Fig 3: Antimicrobial activity of Helicteres isora crude extract, essential oil and antibiotics by agar well diffusion method

4. DISCUSSION:

Methanol extract of H. isora showed the best antimicrobial activity against all tested pathogens. In this therapeutic activity, E.coli showed maximum efficiency in the case of essential oil (figure 3). Malathy et al, showed better efficiency of essential oil on common human wound pathogens which are selected in this research study¹². Various factors affected the antimicrobial activity such as:the extraction process, various places of plant, geographical distribution, and breeding time¹³. Antimicrobial activity of extracts based on the phytochemical and bioactive substances. Similarly, recent work exhibited the presence of phytoconstituents that might be responsible for therapeutic activity (Table 1). Plants Tephrosia and Hibiscus also obtained better existence of phytoconstituents¹⁴.

These activities of H. isora are helpful in the cure of tumors and protect normal cells¹⁵. Thus, therapeutic activities have bioactive substances of the therapeutic plants. Substances were found in known essential oil which is depicted at different retention times and matched from the NIST library and Pubchem (figure 1 and Table 2). Compounds showed the pharmacological properties in drug efficiency are connected to the physicochemical properties to approach ADME prediction of different drugs. It produces the active molecule. In this efficiency, different rules of drugs comply of drug likeliness properties are matched with standard calculations. According to Lipinski's statement, many factors follow various perceptions: number of hydrogen bond donors ≤5 (HBD), molecular weight (MW) ≤500 and number of hydrogen bond acceptors ≤10 (HBA) are useful as oral administration. In this pharmacokinetic study, we checked various parameters for the screening of drugs. Table 3 has shown the result of the absorption ratemeasured by this calculation [(%ABS = 109 – (0.345 ×TPSA)]¹⁶. All compounds were ensued Lipinski rule (Tables 3). All ranges were obtained in-between of 1-2(≤10) of HBA, 1(≤5) of HBD, 1-13(<10) of nROTB demonstrated that all compounds have good druglikeness tendency. Compound 2 and 3b(4) demonstrated all characteristics in a moderate range of pink area. All compounds were found to be in the range of 20.23 -37.30Å² (<140 Å²) of TPSA and also found complete inhibitors of different CYP. Based on these screening, Egg boiled model is demonstrated in Figure2¹⁷. Moreover, Different extracts of this plant are responsible for antimicrobial activities. Similary, Subramanian et al, obtained good potenty of antimicrobial against all these pathogens¹⁸. Raj et al, demonstrated antimicrobial activity of Syzygium caryophyllatum and S. aromaticum of ethanolic extract^19,20,21. Thus, different bioactive compounds revealed good potential of antimicrobial, enter inside the lipid bilayer through the cell surface and finish the antigens^22,23. Shen et al, performed a study of decanoic acid as an antimicrobial improving agent²⁴. Rawat et al, reported various compounds phytol, tetradecanol, n-hexadecanoic acid and pentadecanol²⁵. These compounds exhibited antimicrobial activity against numerous investigated strains, amendable for diarrhea and other treatments.

5. CONCLUSION:

This current study of the fruit of H. isora has scientific assurance which was tested by the various parameters of phytochemical screening, ADME prediction and antimicrobial testing. Moreover, the GCMS chromatogram of essential oil showed the presence of different compounds. Pharmacokinetic studies of ADME demonstrated the highest absorption rate (~100%). All compounds that obey to Lipinski rule indicated good druglikeness properties. Thus, essential oil of this plant exhibited consequences outcomes but not the highest to the limit. This exploration concludes that methanol extract have good potential against all tested pathogens and essential oil have considerable results for further any formulations which are recognized as antimicrobial agents.

6. ACKNOWLEDGMENT:

The authors are heartily thankful to the Head of Department of Botany and Microbiology, Gurukula Kangri Deemed to be University, Haridwar, Uttarakhand.

7. CONFLICT OF INTEREST:

The authors have no conflict of interest related to other findings.

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Received on 02.06.2022 Modified on 04.11.2022

Research J. Pharm. and Tech 2023; 16(11):5015-5020.

DOI: 10.52711/0974-360X.2023.00812