Phytochemical and Pharmacognostic Study of Hedyotis scandens Roxb. from Mizoram, Northeast India

 

Elizabeth Vanlalruati Ngamlai¹, P.C. Lalbiaknii¹, R. Vanlalpeka¹, Vanlalhruaii Ralte²,

F. Lalnunmawia¹*

¹Department of Botany, Mizoram University, Tanhril 796004, India.

²Department of Botany, Pachhunga University College, Aizawl 796001, India.

 

ABSTRACT:

Hedyotis scandens Roxb. (Family Rubiaceae) is used as a medicinal plant in many communities including Mizoram, northeast India. However, there is little evidence of its therapeutic potential, and scientific proof of its antioxidant capacity is  not available to our knowledge and hence, the present study aims to explore the phytoconstituents and curative property of Hedyotis scandens Roxb methanolic leaf extract. Free radical scavenging activity was carried out in vitro. In a concentration-dependent manner, the Hedyotis scandens methanolic leaf extract suppressed the production of ABTS, DPPH and Nitric oxide. The IC₅₀ for DPPH was 35.7±0.14µg/ml, for ABTS it was 50.03±0.12µg/ml and for Nitric oxide the IC₅₀ was 83.61±0.2µg/ml. The presence of compounds such as alkaloid, tannin, flavonoid, and carbohydrate was revealed by phytochemical screening of the plant extract. The total phenolic content was 27.17±0.27 GAE mg/g whereas the total flavonoid content was 29.92±0.3 QE mg/g. The total antioxidant potential of the plant was found to be 98.03±0.14 AE mg/g. Concentration-dependent reducing activity against potassium ferricyanide was seen in the ferric reducing power assay. Antibacterial activity was conducted with two assays and the plant extract showed highest activity against Micrococcus luteus with an inhibition zone of 11.3±0.3mm for 10mg/ml and 15.3±0.3mm for 20mg/ml followed by Escherichia coli at 8.3±0.3mm for 10mg/ml and 10.6±0.3mm, Bacillus subtilis with inhibition zone of 7.3±0.3mm for 10mg/ml and 10.3±0.3mm for 20mg/ml. However, the plant extract did not have any activity against Pseudomonas aeruginosa.

 

 

INTRODUCTION:

Since thousands of years, nature has served as a significant source of medicine.^1,2 Despite long dominance of synthetic drugs in drug discovery and production, bioactive plants still have immense potential in providing new and innovative treatments for therapeutic applications.³ Antioxidant products with fewer side effects are a hot topic in science right now. As a result, it is desirable to create and use more efficient antioxidants of natural origin.⁴ Many antioxidant compounds are synthesized by plants, available in different concentrations and possessing a wide range of chemical and physical properties.⁵

 

Antioxidants are responsible for destroying free radicals, thereby preventing many diseases.^6,7Microbial infections in humans are one of the most serious problems and the most common pathogens causing these infections are micro-organisms like bacteria and fungi. Diseases caused by these microbes are the leading cause of death worldwide.⁸ A rise in multi-resistant strains of clinically significant pathogens has been observed in recent years, which has resulted in an increase in antibiotic-resistant strains.⁹ Plant secondary metabolites can be used as antimicrobial agents in pharmaceutical sciences where the present antibiotics fail to perform their duty due to multidrug resistance.^10,11 Plant extracts, when combined with conventional antibiotics, can enhance antimicrobial activity.^12,13,14,15

 

 

Hedyotis scandens Roxb (family: Rubiaceae) is a type of creeping plant that has several metres long stem, more or less woody, scrambling over the ground or clambering. In Mizoram the plant is called Kelhnamtur or Laikingtuibur and is commonly utilized as a medicinal plant. The indigenous people of Meghalaya use Hedyotis scandens in the treatment of bone fracture, cold and cough, kidney stones, eye problems and sprains.¹⁶ A concoction of Hedyotis scandens is used by the people of Mizoram and ethnic communities of Manipur to treat various ailments including urinary and kidney troubles.^17,18 The plant is also used to treat dysentery and diarrhoae.¹⁹ Therefore, the current study aims in investigating the phytochemical constituents, antibacterial activity and antioxidant potential of Hedyotis scandens leaf extract.

 

MATERIALS AND METHOD:

Plant extract preparation:

Collection of Hedyotis scandens was done from Tanhril, Mizoram (23º44’20.13”N and 92º40’35.78”E) during the month of June-July 2019 and was certified by the Department of Botany, Mizoram University.         Leaves of the plant were cleaned with water and shade dried. The dried plant was then ground to form a rough powder and kept in a sealed storage for future usage.

 

Sample preparation for extraction:

The ground plant material was extracted with a soxhlet apparatus. Extraction was carried out for 72 hours with methanol as solvent. The plant extract was then concentrated and stored at 4°C for future use. Hedyotis scandens methanolic leaf extract will now be termed as HSME.

 

Qualitative phytochemical screening:

Phytochemical investigation had been conducted to find the existence of compounds like alkaloids²⁰, carbohydrates²¹, phytosterols²², tannins^22,23 and glycosides²³, saponin²⁴, flavonoids²⁴, proteins and amino acid²⁴ and reducing sugars²⁴_.

 

Total Phenol Content (TPC) and Total Flavonoid Content (TFC):

HSME’s TPC and TFC were estimated using Folin - Ciocalteu assay^25,26 and Aluminium Chloride method.²⁷ The experiments were conducted thrice for precision and values were expressed.

 

DPPH radical scavenging activity:

DPPH scavenging potential of HSME was evaluated using the method of Leong and Shui.²⁸ The degree of discolouration of DPPH (2, 2- diphenyl-1-picrylhydrazyl) by HSME reflects its scavenging activity.²⁹ The plant extract and standard butylated hydroxytoluene (BHT) was prepared at various concentrations (10 - 100µg/ml). Absorbance was taken at 517nm. The inhibition percentage (I) was determined with the formula:

 

% Inhibition = [(Abs _control – Abs _sample)/Abs _control] × 100

 

ABTS Radical Scavenging Activity:

ABTS assay was carried out based on the proposed method of Re et al with slight modification.³⁰ Different concentrations of HSME and standard BHT were prepared (10 - 100µg/ml). Change in absorbance of the solutions was monitored up to 3 minutes at 745nm. Data was recorded in triad and scavenging potential was determined with the formula:

 

% Inhibition = [(Abs _control – Abs _sample) /Abs _control] × 100

 

Nitric oxide scavenging activity:

Nitric oxide scavenging assay of HSME was estimated with the method of Moon et al.³¹ HSME and standard ascorbic acid were made in different concentrations (10, 20, 40, 60, 80, 100, 150, and 200µg/ml). Absorbance was taken at 550nm using UV-Vis Spectrophotometer. The following formula was used to calculate the scavenging activity:

 

% Inhibition = [(Abs _control – Abs _sample) / Abs _control] × 100

 

Determination of Total Antioxidant Activity:

The method of Prieto et al was employed to estimate phosmomolybdate.³² A series of concentrations of standard ascorbic acid were prepared (10 - 100µg/ml). A standard curve was created by measuring absorbance at 695nm with a UV-Vis spectrophotometer. The same procedure was used for HSME from a stock solution of 1mg/ml. Total antioxidant activity of HSME was then expressed in milligrams of ascorbic acid equivalent per gram of dried extract.

 

Determination of Reducing Power:

Reducing power of HSME was calculated using the method of Oyaizu with ascorbic acid as standard.³³ Different concentrations (10 - 100µg/ml) of HSME and ascorbic acid were prepared. Absorbance was taken at 700nm. Increase in absorbance meant increased reducing power.

 

Antimicrobial activity:

a. Microorganisms:

Two gram positive bacteria Bacillus subtilis (ATCC- 11774) and Micrococcus luteus (ATCC- 10240) and two gram negative bacteria Escherichia coli (ATCC-10536) and Pseudomonas aeruginosa (ATCC-10145) were used for the current study. These microorganisms were procured from the Institute of Microbial Technology (IMTECH), Chandigarh, Punjab, India. Prior to the experiment, the microorganisms were sub cultured in nutrient broth and incubated at 37°C for 24 hours.

 

b. Disc diffusion method:

Antimicrobial activity of the plant extract was tested on the test microorganisms by disc diffusion method.^{34, 35} Two concentrations namely 10mg/ml and 20mg/ml of HSME was loaded on to the sterile paper disc 6mm in diameter. 100µg of ceftriaxone was used as standard. The control experiment was maintained containing the bacteria culture only. 100µl of 0.5 McFarland standardized bacterial cultures were inoculated on the solidified Mueller Hinton Agar media, and discs containing the plant extract were then placed on the top of solidified media. The plates were incubated at 37°C for 12 hrs. After incubation, the diameter of the zone of growth inhibition was observed and recorded, and then compared with that of the standard antibiotic.

 

c. Minimum Inhibitory Concentration (MIC):

MIC of the plant extract was determined on sterile 1.2ml 96-well plates using resazurin as an indicator of bacterial viability with slight modification.^36,37 Resazurin was prepared as 10g/l sterile water stock solution and diluted at 1: 10 in sterile water when required. The wells were filled with 0.5ml of sterilised Mueller Hinton Broth, followed by 0.5ml of HSME at various doses. Following that, 0.1ml (5 × 10⁸ cfu/mL) of the bacterial inoculum was added to each well and carefully mixed. The plates were incubated in at 37ºC for 12 hrs. After 12 hrs of incubation 10µl of resazurin was added and then incubated again for another 3-4hr. Colour change in the wells was monitored visually. Concentrations with pink colour after the incubation period in the modified resazurin assay indicated growth of the bacteria while plates with blue signify no growth. This assay is helpful to screen the natural product bioassay both in vitro and in vivo, cost effective rapid and easy to perform.³⁸

 

RESULTS AND DISCUSSION

Phytochemical screening, Total Phenolic Content (TPC) and Total Flavonoid Content (TFC):

 

Table 1. Phytochemical screening of HSME

Sl. No	Compounds	Phytochemical test	Present/Absent
1	Alkaloids	Mayer’s test	Present
		Dragendroff’s test	Present
		Wagner’s test	Present
		Hager’s test	Present
2	Carbohydrates	Molisch’s test	Present
		Benedict’s test	Present
		Fehling’s test	Present
3	Phytosterols	Leibermann-Burchard’s test	Absent
		Salkwoski reaction	Absent
4	Glycosides	Legal’s test	Absent
		Keller Killiani’s test	Absent
5	Tannin	FeCl3	Present
		K2Cr3O7	Absent
		Lead acetate test	Absent
6	Saponin	Foam of froth test	Absent
7	Flavonoid	Zinc-Hydrochloride reduction test	Present
8	Proteins and amino acid	Xanthoproteic test	Absent
		Hydrolysis test	Absent

 

 

Fig. 1&2: Standard curve of gallic acid for total phenol assay and Standard curve of quercetin for total flavonoid assay. Dotted line represents the linear graph. (Values are in mean ± SEM, n = 3).

 

Phytochemical analysis of HSME revealed the presence of alkaloids, carbohydrate, tannin and flavonoid while saponin, phytosterols, protein and amino acid were found to be absent. Using the gallic acid standard graph, TPC was calculated as milligrams of gallic acid equivalent of the dried extract (Fig.1). TPC of HSME was 27.17±0.27 GAE mg/g. Using quercetin as a standard, the TFC was estimated for the plant (Fig. 2). The value is given in milligrams of quercetin per gram of the dried extract. According to the standard graph the plant extract contained 29.92±0.3 QE mg/g. The presence of phenolic and flavonoid compounds in HSME was however revealed by quantitative phytochemical analysis. Phytochemicals, found in plants, have considerable health benefits for humans.³⁹ Five novel phenolic glycosides were identified from Hedyotis scandens in compliance with this investigation.⁴⁰Phenols and flavonoids are antioxidants that scavenge free radicals engaged in oxidative processes through hydrogenation or complexing with oxidising agents and thereby help in the prevention and treatment of various diseases.^{41, 42}

 

DPPH Radical Scavenging Activity:

 

Fig. 3: DPPH scavenging activity of HSME and Standard BHT. The percentage of inhibition is plotted against concentration of sample. Values are expressed as Mean ± SEM, n= 3

Figure.3 shows a rise in the DPPH scavenging activity of HSME with rise in concentration as evidenced by the increasing discoloration of DPPH. Standard BHT demonstrated greater scavenging action than the extract. The IC₅₀ of the standard BHT was 17.29±0.13 µg/ml whereas the IC₅₀ of the extract was 35.77±0.14 µg/ml. HSME effectively reduced the stable radical DPPH to the yellow coloured diphenyl – pycrylhydrazine. This might be due to the presence of hydrogen donating properties of compounds such as ascorbic acid, glutathione, tocopherol, and polyhydroxyl aromatic compounds in HSME.⁴³In vitro studies have demonstrated that other species of Hedyotis also successfully scavenged DPPH free radicals.⁴⁴

 

ABTS Radical Scavenging Activity:

 

Fig. 4: ABTS scavenging activity of HSME and Standard BHT. The percentage of inhibition is plotted against concentration of sample. Values are expressed as Mean ± SEM, n= 3

 

HSME's ABTS + radical scavenging activity increased in a concentration dependent manner, as proved by the discoloration of ABTS + with increasing concentration which was measured spectrophotometrically at 745nm (Fig. 4) The IC₅₀ of the plant extract was 50.03±0.12 µg/ml while the IC₅₀ of the standard BHT was calculated to be 8.37±0.21 µg/ml. Hedyotis corymbosa methanolic extract has also been shown to successfully scavenge ABTS^.+.⁴⁵ The efficiency of suppressing the radical is determined by the molecular weight of phenolic compounds, amount of aromatic rings, and type of hydroxyl group substitution rather than the specific functional group.⁴⁶ Based on this study, we found that HSME has potent antioxidant properties and can effectively scavenge ABTS.+ which can be attributed to its phenolic compounds and flavonoids. Different types of phenolic compounds will exhibit different reactivity toward ABTS, which could account for differences in the scavenging potential of the extract and standard BHT.⁴⁷

 

Nitric oxide Scavenging Activity:

 

Fig. 5: Nitric oxide scavenging activity of HSAE and Standard Ascorbic Acid. The percentage of inhibition is plotted against concentration of sample. Values are expressed as Mean±SEM, n= 3.

 

HSME’s nitric oxide scavenging activity was concentration dependent (Fig. 5). However, HSME demonstrated a lower scavenging activity than the standard ascorbic acid at all concentrations. IC₅₀ for the standard, calculated from the graph was found to be 64.2±0.14 µg/ml and the IC₅₀ of HSME was 83.61±0.2 µg/ml. Mamallian cells produce nitric oxide, a free radical that is involved in a variety of physiological processes However, production of excess nitric oxide leads to a  number of diseases.⁴⁸ The nitric oxide scavenging properties of other species of Hedyotis have been reported.^49,45 Flavonoids and phenolic compounds have been reported to have nitric oxide-scavenging properties.^50-54 Therefore, HSME's ability to scavenge nitric oxides can be attributed to these compounds.

 

Total Antioxidant Activity:

 

Fig. 6: Standard curve of Ascorbic acid for Total Antioxidant Assay. The percentage of inhibition is plotted against concentration of sample. Values are expressed as Mean±SEM, n= 3.

 

The phosphomolybdenum assay was used to calculate the total antioxidant activity of HSME. The value was given in terms of ascorbic acid equivalent (AE) per gram of dried extract (Fig. 6). HSME showed 98.03±0.14 AE mg/g of antioxidant activity. This assay relies on the reduction of molybdenum (IV) to molybdenum (V) by the plant sample and the production of phosphate/molybdenum (V) complex at acidic conditions, which is green in colour. Flavonoid and polyphenols found in medicinal plants are responsible for the plant's phosphomolybdate scavenging activity. ^{55, 56} Therefore, the presence of phenolics and flavonoids in HSME may have contributed to its total antioxidant capacity.

 

Reducing power:

 

Fig. 7: Reducing power of HSME and Standard Ascorbic Acid. The percentage of inhibition is plotted against concentration of sample. Values are expressed as Mean ± SEM, n= 3.

 

HSME reduced potassium ferricyanide in a concentration dependent manner as seen in Fig 7. Standard ascorbic acid showed high activity while Hedyotis scandens extract showed only minimal activity. Reduced Fe³⁺ is a measure of electron donating activity, one of the main functions of phenolic antioxidants.⁵⁷ A compound's reducing ability is mediated by reductones, which contribute a hydrogen atom to break the free radical chain, resulting in antioxidant activity.⁵⁸ Antioxidants present in the plant reduce the Fe³⁺/Ferricyanide complex to the Fe²⁺/Ferrous form.^59,60 HSME's reducing power indicates that it contributes significantly to the antioxidant effect.

 

Antibacterial activity and MIC.

The antibacterial activity and MIC of HSME is shown in Table 2. HSME exhibited maximum action against Micrococcus luteus (ATCC-10240) followed by Escherichia coli (ATCC-10536). In comparison to the other two bacteria, HSME exhibited the lowest activity against Bacillus subtilis (ATCC-11774), which could be attributed to their ability to develop endospores which are more resistant to environmental conditions than the other studied bacteria.^61,62 HSME did not show any activity against Pseudomonas aeruginosa (ATCC-10145) at both the concentrations. On the other hand standard ceftriaxone, a broad spectrum antibiotic was found to be most active against Bacillus subtilis (ATCC-11774) and least active against Escherichia coli (ATCC-10536).

 

Table2. Antimicrobial activity and MIC of HSME. Values are expressed as Mean ± SEM, n= 3.

	10 (mg/ml)	20 (mg/ml)	Ceftriaxone (10µg/ml)	MIC (mg/ml)
		Zone of inhibition (mm)
Micrococcus luteus (ATCC-10240)	11.3 ± 0.3mm	15.3 ± 0.3mm	24 ± 0.01mm	6
Escherichia coli (ATCC-10536)	8.3 ± 0.3mm	10.6 ± 0.3mm	22 ± 0.3mm	8
Bacillus subtilis (ATCC- 11774)	7.3 ± 0.3mm	10.3 ± 0.3mm	25 ± 0.03mm	10

 

The MIC test also showed that HSME was most effective against Micrococcus luteus followed by Escherichia coli and Bacillus subtilis. To find the MIC value, we took lowest concentration of the extract in which color change occurred. All experiments were conducted in triplicate. All the bacteria used for this study are opportunistic pathogens and have been reported to cause various diseases.^{63,6465,66,67} In line with our study, another study had found that the ethanolic extract of the H. scandens had strong antibacterial activity against K. pneumonia in a concentration dependent manner.⁶⁸ Studies have linked secondary metabolites from plants with antimicrobial properties. These compounds protect the plants against bacterial, viral and fungal infection.^69,70,71 Therefore, the antibacterial activity of HSME can be due to the presence of secondary compounds in the plant as confirmed from the present study.

 

CONCLUSION:

An analysis of the methanolic extract of Hedyotis scandens leaves demonstrated the presence of significant quantities of bioactive compounds and these compounds are responsible for the antibacterial and free radical scavenging activity of the plant. Our study shows that the plant has scavenging potential, antioxidant capacity, reducing properties and antibacterial activity which suggest that it might serve as a natural alternative to synthetic drugs.

 

ACKNOWLEDGMENT:

The authors would like to express their gratitude to the Department of Botany and Zoology, Pachhunga University College Campus, Mizoram University, Aizawl, Mizoram, for providing the necessary facilities for the experiments.

 

CONFLICT OF INTEREST:

There is no conflict of interest among the authors.

 

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Accepted on 27.03.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(12):5483-5489.